Targeted binding agents against B7-H1

ABSTRACT

Human monoclonal antibodies directed against B7-H1 and uses of these antibodies in diagnostics and for the treatment of diseases associated with the activity and/or expression of B7-H1 are disclosed. Additionally, hybridomas or other cell lines expressing such antibodies are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/333,683, filed Oct. 25, 2016, said application Ser. No. 15/333,683 isa Continuation of U.S. application Ser. No. 14/271,108, filed on May 6,2014, now U.S. Pat. No. 9,493,565, issued Nov. 15, 2016, said U.S.application Ser. No. 14/271,108 is a Continuation of U.S. applicationSer. No. 13/511,538 filed on Aug. 7, 2012, now U.S. Pat. No. 8,779,108issued Jul. 15, 2014, said application Ser. No. 13/511,538 is a U.S.National Stage Application of International Application No.PCT/US2010/058007 filed on Nov. 24, 2010, said International ApplicationNo. PCT/US2010/058007 claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/264,061 filed Nov. 24, 2009. Each of theabove listed applications is incorporated by reference herein in itsentirety for all purposes.

REFERENCE TO THE SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 17, 3019 isnamed B7H1-100US2_SL.txt and is 60,061 bytes in size.

FIELD OF THE INVENTION

The invention relates to targeted binding agents against the B7-H1protein and uses of such agents. In some embodiments, the inventionrelates to fully human monoclonal antibodies directed to B7-H1 and usesof these antibodies. Aspects of the invention also relate to cell linesexpressing such targeted binding agents or antibodies. The describedtargeted binding agents are useful as diagnostics and for the treatmentof diseases associated with the activity and/or expression of B7-H1.

DESCRIPTION OF THE RELATED ART

An adaptive immune response involves activation, selection, and clonalproliferation of 30 two major classes of lymphocytes termed T cells andB cells. After encountering an antigen, T cells proliferate anddifferentiate into antigen-specific effector cells, while B cellsproliferate and differentiate into antibody-secreting cells. T cellactivation is a multi-step process requiring several signaling eventsbetween the T cell and an antigen-presenting cell (APC). For T cellactivation to occur, two types of signals must be delivered to a restingT cell. The first type is mediated by the antigen-specific T cellreceptor (TcR), and confers specificity to the immune response. Thesecond, costimulatory, type regulates the magnitude of the response andis delivered through accessory receptors on the T cell.

A primary costimulatory signal is delivered through the activating CD28receptor upon engagement of its ligands B7-1 or B7-2. In contrast,engagement of the inhibitory CTLA-4 receptor by the same B7-1 or B7-2ligands results in attenuation of a T cell response. Thus, CTLA-4signals antagonize costimulation mediated by CD28. At high antigenconcentrations, CD28 costimulation overrides the CTLA-4 inhibitoryeffect. Temporal regulation of the CD28 and CTLA-4 expression maintainsa balance between activating and inhibitory signals and ensures thedevelopment of an effective immune response, while safeguarding againstthe development of autoimmunity.

Molecular homologues of CD28 and CTLA-4 and their B-7 like ligands havebeen recently identified. ICOS is a CD28-like costimulatory receptor.PD-1 (Programmed Death 1) is an inhibitory receptor and a counterpart ofCTLA-4. This disclosure relates to modulation of immune responsesmediated by B7-H1.

B7-H1, also known as PD-L1, is a type I transmembrane protein ofapproximately 53 kDa in size. In humans B7-H1 is expressed on a numberof immune cell types including activated and anergic/exhausted T cells,on naïve and activated B cells, as well as on myeloid dendritic cells(DC), monocytes and mast cells. It is also expressed on non-immune cellsincluding islets of the pancreas, Kupffer cells of the liver, vascularendothelium and selected epithelia, for example airway epithelia andrenal tubule epithelia, where its expression is enhanced duringinflammatory episodes. B7-H1 expression is also found at increasedlevels on a number of tumours including, but not limited to breast,colon, colorectal, lung, renal, including renal cell carcinoma, gastric,bladder, non-small cell lung cancer (NSCLC), hepatocellular cancer(HCC), and pancreatic cancer, as well as melanoma.

B7-H1 is a member of the B7 family of proteins, which contain twoextracellular Ig domains, one N-terminal V-type domain followed by aC-type domain. The intracellular domain of 30 amino acids lengthcontains no obvious signaling motifs, but does bear a potential site forprotein kinase C phosphorylation. The murine form of B7-H1 bears 69%amino acid identity with the human form of B7-H1, and also shares aconserved structure.

B7-H1 is known to bind two alternative ligands, the first of these,PD-1, is a 50-55 kDa type I transmembrane receptor that was originallyidentified in a T cell line undergoing activation-induced apoptosis.PD-1 is expressed on activated T cells, B cells, and monocytes, as wellas other cells of the immune system and binds both B7-H1 (PD-L1) and therelated B7-DC (PD-L2). The second is the B7 family member B7-1, which isexpressed on activated T cells, B cells, monocytes and antigenpresenting cells.

PD-1 is a member of the immunoglobulin (Ig) superfamily that contains asingle Ig V-like domain in its extracellular region. The PD-1cytoplasmic domain contains two tyrosines, with the mostmembrane-proximal tyrosine (VAYEEL in mouse PD-1) located within an ITIM(immuno-receptor tyrosine-based inhibitory motif). The presence of anITIM on PD-1 indicates that this molecule functions to attenuate antigenreceptor signaling by recruitment of cytoplasmic phosphatases. Human andmurine PD-1 proteins share about 60% amino acid identity withconservation of four potential N-glycosylation sites, and residues thatdefine the Ig-V domain.

The ITIM in the cytoplasmic region and the ITIM-like motif surroundingthe carboxy-terminal tyrosine (TEYATI in human and mouse) are alsoconserved between human and murine orthologues.

Signalling via the PD-1/B7-H1 axis is believed to serve critical,non-redundant functions within the immune system, by negativelyregulating T cell responses. This regulation is involved in T celldevelopment in the thymus, in regulation of chronic inflammatoryresponses and in maintenance of both peripheral tolerance and immuneprivilege. The critical nature of these functions is exemplified inPD-1-deficient mice, which exhibit an autoimmune phenotype. PD-1deficiency in the C57BL/6 mice results in chronic progressive lupus-likeglomerulonephritis and arthritis. In Balb/c mice, PD-1 deficiency leadsto severe cardiomyopathy due to the presence of heart-tissue-specificself-reacting antibodies. The function of signaling via B7-H1/B7-1 isless clear, but is thought to also be involved in delivering negativeregulatory signals to both T cells and antigen presenting cells.

B7-H1 expression on tumour cells is believed to aid tumours in evadingdetection and elimination by the immune system. B7-H1 functions in thisrespect via several alternative mechanisms including driving exhaustionand anergy of tumour infiltrating T lymphocytes, stimulating secretionof immune repressive cytokines into the tumour micro-environment,stimulating repressive regulatory T cell function and protecting B7-H1expressing tumour cells from lysis by tumour cell specific cytotoxic Tcells.

In general, a need exists to provide safe and effective therapeuticmethods for disorders associated with repression of an immune responsesuch as, for example cancer and chronic viral infection. Modulation ofthe immune responses involved in these disorders can be accomplished bymanipulation of the B7-H1/PD-1 pathway.

SUMMARY OF THE INVENTION

The present invention relates to targeted binding agents thatspecifically bind to B7-H1 and inhibit the biological activity of B7-H1.In one embodiment of the invention, the invention relates to targetedbinding agents that specifically bind to B7-H1 and thereby inhibit B7-H1activity. In another embodiment of the invention, the invention relatesto targeted binding agents that specifically bind to B7-H1 and therebyinhibit binding of B7-H1 to PD-1. In yet another embodiment of theinvention, the invention relates to targeted binding agents that blockB7-H1 induced T-cell suppression and thereby enhance anti-tumorimmunity. In yet another embodiment of the invention, the inventionfurther relates to targeted binding agents that can further stimulateone or more of the following activities including T cell proliferation,IFN-γ and/or IL-2 secretion in mixed lymphocyte reactions. Embodimentsof the invention relate to targeted binding agents that specificallybind to B7-H1 and inhibit the biological activity of B7-H1. In oneembodiment the targeted binding agent inhibits at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% of the biological activity than wouldoccur in the absence of the targeted binding agent.

Embodiments of the invention relate to targeted binding agents thatspecifically bind to B7-H1 and thereby inhibit B7-H1 activity. In oneembodiment the targeted binding agent inhibits at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% of B7-H1 activity than would occur inthe absence of the targeted binding agent.

Embodiments of the invention relate to targeted binding agents thatspecifically bind to B7-H1 and thereby inhibit binding to PD-1. In oneembodiment the targeted binding agent inhibits at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% of B7-H1/PD-1 receptor ligand bindingcompared to what would occur in the absence of the targeted bindingagent.

In another embodiment, the targeted binding agents of the invention caninhibit binding of PD-1/Fc to human B7-H1 expressed on ES-2 cells. Inone embodiment the targeted binding agent inhibits binding with an IC50of less than 1 nM, 0.5 nM, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07 or 0.06nM. Further, in another embodiment, the antibodies of the invention havean IC50 of about 1 nM down to about 0.06 nM; or of about 0.5 nM down toabout 0.06 nM; or of about 0.1 nM down to about 0.06 nM; or of about 1nM down to about 0.1 nM; or of about 1 nM down to about 0.5 nM.

Embodiments of the invention relate to targeted binding agents thatspecifically bind to B7-H and thereby inhibit binding to its ligandB7-1. In one embodiment the targeted binding agent inhibits at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, 95%, 96%, 97%, 98% 99% or 100% of B7-H1/B7-1receptor ligand binding compared to what would occur in the absence ofthe targeted binding agent.

Embodiments of the invention relate to targeted binding agents thatspecifically bind to B7-H1 and inhibit B7-H1 induced tumorproliferation. In one embodiment the targeted binding agent inhibits atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% of B7-H1 inducedtumor proliferation than would occur in the absence of the targetedbinding agent.

Further embodiments of the invention relate to targeted binding agentsthat specifically bind to B7-H1 and thereby inhibit B7-H1 induced tumorcell survival. In one embodiment the targeted binding agent inhibits atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% of B7-H1 inducedtumor cell survival than would occur in the absence of the targetedbinding agent.

Further embodiments of the invention relate to targeted binding agentsthat specifically bind to B7-H1 and thereby inhibit tumour growth ofA375 or HPAC cancer cell lines. In one embodiment the targeted bindingagent inhibits at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% ofgrowth of cancer cells at day 30 compared to an isotype control.

Further embodiments of the invention relate to targeted binding agentsthat specifically bind to B7-H1 and thereby inhibit B7-H1 mediatedsuppression of tumour reactive T-cells, thereby enhancing anti-tumourcytolytic T-cell activity. In one embodiment the targeted binding agentinhibits at least 5%, at least 10%, at least 15%, at least 200, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95% of B7-H1mediated suppression of tumour reactive T-cell activity than would occurin the absence of the targeted binding agent.

Further embodiments of the invention relate to targeted binding agentsthat specifically bind to B7-H1 and thereby enhance anti-tumourimmunity. In one embodiment the targeted binding agent enhances at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 700, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% of anti-tumor immunitythan would occur in the absence of the targeted binding agent.

Further embodiments of the invention relate to targeted binding agentsthat specifically bind to B7-H1 and thereby inhibit cell proliferation.In one embodiment the targeted binding agent inhibits at least 5%, atleast 10%, at least 15%, at least 200, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% of cell proliferation thanwould occur in the absence of the targeted binding agent.

Further embodiments of the invention relate to targeted binding agentsthat specifically bind to B7-H1 and increase specific cytolytic (CTL)activity against B7-H1 expressing tumor cells. In one embodiment, theantibodies of the invention have an EC50 of less than or equal to 100nM, 50 nM or 1 nM. Further, in another embodiment, the antibodies of theinvention have an EC50 of about 100 nM down to about 1 nM; or of about50 nM down to about 1 nM; or of about 20 nM down to about 1 nM; or ofabout 100 nM down to about 50 nM; or of about 100 nM down to about 70nM.

Further embodiments of the invention relate to targeted binding agentsthat specifically bind to B7-H1 and inhibit B7-H1 mediated suppressionof T-cell proliferation at an EC50 less than or equal to 100 nM. In oneembodiment, the antibodies of the invention have an EC50 of less than orequal to 100 nM, e.g., 90, 80, 70, 60, 50, 40, 30, 20 or 10 nM. Further,in another embodiment, the antibodies of the invention have an EC50 ofabout 100 nM down to about 10 nM: or of about 50 nM down to about 10 nM;or of about 20 nM down to about 10 nM; or of about 100 nM down to about50 nM; or of about 100 nM down to about 70 nM; or of about 100 nM downto about 80 nM.

The targeted binding agents also inhibit tumour cell adhesion, motility,invasion and cellular metastasis and in addition, the targeted bindingagents are useful for reducing tumour growth. Mechanisms by which thiscan be achieved can include, and are not limited to, inhibiting B7-H1activity.

In one embodiment of the invention, the targeted binding agent is anantibody. In one embodiment of the invention, the targeted binding agentis a monoclonal antibody. In one embodiment of the invention, thetargeted binding agent is a fully human monoclonal antibody or afragment thereof. Such monoclonal antibodies may be referred to hereinas anti-B7-H1 antibodies or antibodies of the invention.

Antibodies, monoclonal antibodies and human monoclonal antibodiesinclude the antibodies of the IgG1, IgG2, IgG3 and IgG4 isotypes, forexample IgG2. In one embodiment of the invention, the targeted bindingagent is a fully human monoclonal antibody of the IgG2 isotype. Thisisotype has reduced potential to elicit effector function in comparisonwith other isotypes, which may lead to reduced toxicity. In anotherembodiment of the invention, the targeted binding agent is a fully humanmonoclonal antibody of the IgG1 isotype. The IgG1 isotype has increasedpotential to elicit Antibody Directed Cell-mediated Cytotoxicity (ADCC)in comparison with other isotypes, which may lead to improved efficacy.The IgG1 isotype has improved stability in comparison with otherisotypes, e.g. IgG4, which may lead to improved bioavailability/ease ofmanufacture/longer half-life. In one embodiment, the fully humanmonoclonal antibody of the IgG1 isotype is of the z, za or f allotype.In one embodiment of the invention, the targeted binding agent hasdesirable therapeutic properties, selected from one or more of thefollowing: high binding affinity for B7-H1, the ability to inhibit B7-H1activity in vitro and in vivo, the ability to inhibit B7-H1-mediatedtumour cell survival, and the ability to inhibit B7-H1 mediatedsuppression of tumour reactive T-cells, which may in turn reduce tumourcell proliferation, motility, invasion, metastasis, and tumour growth.

In one embodiment, the invention includes antibodies that specificallybind to B7-H1 with very high affinities (Kd). In some embodiments of theinvention, the targeted binding agent binds B7-H1 with a bindingaffinity (Kd) of less than 5 nanomolar (nM). In other embodiments, thetargeted binding agent binds with a Kd of less than 4 nM, 3 nM, 2.5 nM,2 nM or 1 nM. Further, in some other embodiments antibodies of theinvention binds B7-H1 with a Kd of about 5 nM to about 1 nM; or about 5nM to about 2 nM; or about 5 nM to about 3 nM; or about 5 nM to about 4nM; or about 3 nM to about 1 nM; or about 2 nM to about 1 nM. In someembodiments of the invention, the targeted binding agent binds B7-H1with a Kd of less than 950 picomolar (pM). In some embodiments of theinvention, the targeted binding agent binds B7-H1 with a Kd of less than900 pM. In other embodiments, the targeted binding agent binds B7-H1with a Kd of less than 800 pM, 700 pM or 600 pM. In some embodiments ofthe invention, the targeted binding agent binds B7-H1 with a Kd of lessthan 500 pM. In other embodiments, the targeted binding agent bindsB7-H1 with a Kd of less than 400 pM. In still other embodiments, thetargeted binding agent binds B7-H1 with a Kd of less than 300 pM. Insome other embodiments, the targeted binding agent binds B7-H1 with a Kdof less than 200 pM. In some other embodiments, the targeted bindingagent binds B7-H1 with a Kd of less than 100 pM. Further, in some otherembodiments antibodies of the invention binds B7-H1 with a Kd of about900 pM to about 100 pM; or about 900 pM to about 200 pM; or about 900 pMto about 300 pM; or about 900 pM to about 400 pM; or about 900 pM toabout 500 pM; or about 900 pM to about 600 pM; or about 900 pM to about700 pM; or about 200 pM to about 100 pM; or about 300 pM to about 200pM; or about 400 pM to about 300 pM. In some other embodiments, thetargeted binding agent binds B7-H1 with a Kd of less than 90 pM, 80 pM,70 pM, 60 pM, 55 pM or 50 pM. In some other embodiments, the targetedbinding agent binds B7-H1 with a Kd of less than 60 pM. In some otherembodiments, the targeted binding agent binds B7-H1 with a Kd of lessthan 55 pM. Further, in some other embodiments antibodies of theinvention binds B7-H1 with a Kd of about 100 pM to about 50 pM; or about100 pM to about 70 pM; or about 100 pM to about 80 pM; or about 100 pMto about 90 pM; or about 70 pM to about 50 pM; or about 60 pM to about50 pM; or about 55 pM to about 50 pM. The Kd may be assessed using amethod described herein or known to one of skill in the art (e.g., aBIAcore assay, ELISA) (Biacore International AB, Uppsala, Sweden).Targeted binding agents of the invention have considerably improvedbinding affinities for B7-H1 in comparison with the antibodies reportedin the prior art.

The binding properties of the targeted binding agent or antibody of theinvention may also be measured by reference to the dissociation orassociation rates (k_(off) and k_(on) respectively).

In one embodiment of the invention, a targeted binding agent or anantibody of the invention may have a k_(on) rate (antibody (Ab)+antigen(Ag)^(k) ^(on) →Ab−Ag) of at least 10⁴ M⁻¹s⁻¹, at least 5×10⁴ M⁻¹s⁻¹, atleast 10⁵ M⁻¹s⁻¹, at least 2×10⁵ M⁻¹s⁻¹, at least 5×10⁵ M⁻¹s⁻¹, at least10⁶ M⁻¹s⁻¹, at least 5×10⁶ M⁻¹s⁻¹, at least 10⁷ M⁻¹s⁻¹, at least 5×10⁷M⁻¹s⁻¹, or at least 10⁸ M⁻¹s⁻¹ as measured by a BIAcore assay. Further,in some other embodiments antibodies of the invention have a k_(on) rateof about 5×10⁴ M⁻¹s⁻¹ to about 5×10⁸ M⁻¹s⁻¹; or of about 5×10⁵ M⁻¹s⁻¹ toabout 5×10⁸ M⁻¹s⁻¹; or of about 5×10⁶ M⁻¹s⁻¹ to about 5×10⁸ M⁻¹s⁻¹; orof about 5×10⁷ M⁻¹s⁻¹ to about 5×10⁸ M⁻¹s⁻¹, as measured by a BIAcoreassay.

In another embodiment of the invention, targeted binding agent or anantibody may have a k_(off) rate ((Ab−Ag)^(k) ^(off) →antibody(Ab)+antigen (Ag)) of less than 5×10⁻¹s⁻¹, less than 10⁻¹ s⁻¹, less than5×10⁻² s⁻¹, less than 10⁻² s⁻¹, less than 5×10⁻³ s⁻¹, less than 10⁻³s⁻¹, less than 5×10⁻⁴ s⁻¹, less than 10⁻⁴ s⁻¹, less than 5×10⁻⁵ s⁻¹,less than 10⁻⁵ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁶ s⁻¹, less than5×10⁻⁷ s⁻¹, less than 10⁻⁷ s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁸s⁻¹, less than 5×10⁻⁹ s⁻¹, less than 10⁻⁹ s⁻¹, or less than 10¹⁰ s⁻¹ asmeasured by a BIAcore assay. Further, in some other embodimentsantibodies of the invention have a k_(off) rate of about 1×10⁻⁴ s⁻¹ toabout 1×10⁻⁵ s⁻¹; or of about 1×10⁻⁴ s⁻¹ to about 5×10⁻⁴ s⁻¹, asmeasured by a BIAcore assay.

The targeted binding agent of the invention specifically binds humanB7-H1. In some examples, the targeted binding agent of the inventiondoes not bind other immune co-modulatory proteins, e.g., human PD-L2,human B7-H2, human B7-H3, human CD28, human CTLA-4 and human PD1.

In another embodiment, the targeted binding agent of the invention iscross-reactive with other B7-H1 proteins from other species. In oneembodiment, the targeted binding agent of the invention iscross-reactive with cynomolgus monkey B7-H1. In another embodiment, thetargeted binding agent of the invention is cross-reactive with mouseB7-H1, e.g., 2.7A4. In another embodiment, the targeted binding agent ofthe invention is cross-reactive with cynomolgus monkey B7-H1 and withmouse B7-H1, e.g., 2.7A4. In another embodiment, the targeted bindingagent of the invention is cross-reactive with cynomolgus monkey B7-H1but not with mouse B7-H1, e.g., 2.9D10 and 2.14H9.

In one embodiment, the targeted binding agent or antibody comprises asequence comprising any one of the heavy chain sequences (VH) shown inTable 8. In another embodiment the targeted binding agent or antibodycomprises a sequence comprising any one of the heavy chain sequences ofantibodies 2.9D10, 2.7A4, 2.14H9, 3.15G8, 2.20A8, 3.18G1, 2.7A4OPT, or2.14H9OPT. Light-chain promiscuity is well established in the art, thus,a targeted binding agent or antibody comprising a sequence comprisingany one of the heavy chain sequences of antibodies 2.9D10, 2.7A4,2.14H9, 3.15G8, 2.20A8, 3.18G1, 2.7A4OPT, or 2.14H9OPT, or anotherantibody as disclosed herein, may further comprise any one of the lightchain sequences (VL) shown in Table 9 or of antibodies 2.9D10, 2.7A4,2.14H9, 3.15G8, 2.20A8, 3.18G1, 2.7A4OPT or 2.14H9OPT, or other antibodyas disclosed herein. In another embodiment the targeted binding agent orantibody comprises a sequence comprising any one of the heavy chainsequences of antibodies 2.9D10, 2.7A4, 2.14H9, 3.15G8, 2.20A8, 3.18G1,2.7A4OPT, or 2.14H9OPT and further comprising the corresponding lightchain sequence of antibody 2.9D10, 2.7A4, 2.14H9, 3.15G8, 2.20A8,3.18G1, 2.7A4OPT, or 2.14H9OPT. In some embodiments, the antibody is afully human monoclonal antibody.

In one embodiment, the targeted binding agent or antibody comprises asequence comprising any one of the light chain sequences shown in Table9. In another embodiment, the targeted binding agent or antibodycomprises a sequence comprising any one of the light chain sequences ofantibodies 2.9D10, 2.7A4, 2.14H9, 3.15G8, 2.20A8, 3.18G1, 2.7A4OPT, or2.14H9OPT. In some embodiments, the antibody is a fully human monoclonalantibody.

In another embodiment the targeted binding agent or antibody comprises asequence comprising any of the heavy chain sequence of antibody 2.7A4and further comprising the light chain sequence of antibody 2.7A4. Inanother embodiment the targeted binding agent or antibody comprises asequence comprising any of the heavy chain sequence of antibody 2.14H9and further comprising the light chain sequence of antibody 2.14H9. Inanother embodiment the targeted binding agent or antibody comprises asequence comprising any of the heavy chain sequence of antibody 2.9D10and further comprising the light chain sequence of antibody 2.9D10. Inanother embodiment the targeted binding agent or antibody comprises asequence comprising any of the heavy chain sequence of antibody2.7A.4OPT and further comprising the light chain sequence of antibody2.7A.4OPT. In another embodiment the targeted binding agent or antibodycomprises a sequence comprising any of the heavy chain sequence ofantibody 2.14H9OPT and further comprising the light chain sequence ofantibody 2.14H9OPT.

In some embodiments, the targeting binding agent is any one of themonoclonal antibodies as shown in Table 1. In some embodiments, thetargeting binding agent is a monoclonal antibody selected from the groupconsisting of: 2.7A4, 2.14H9, 2.9D10, 2.7A4OPT or 2.14H9OPT. In oneembodiment, the targeted binding agent comprises one or more of fullyhuman monoclonal antibodies 2.7A4, 2.14H9, 2.9D10, 2.7A4OPT or2.14H9OPT. In certain embodiments, the targeting binding agent ismonoclonal antibody 2.7A4. In certain other embodiments, the targetingbinding agent is monoclonal antibody 2.14H9. In still other embodiments,the targeting binding agent is monoclonal antibody 2.9D10. In certainembodiments, the targeting binding agent is monoclonal antibody2.7A4OPT. In certain other embodiments, the targeting binding agent ismonoclonal antibody 2.14H9OPT. In additional embodiments, the targetedbinding agent is derivable from any of the foregoing monoclonalantibodies.

In another embodiment the targeted binding agent may comprise a sequencecomprising any one of the CDR1, CDR2 or CDR3 of the heavy chain variablesequences encoded by a polynucleotide in a plasmid designated 2.7A4_G,2.14H9_G, and 2.9D10_NG which were deposited at NCIMB under number 41598on Nov. 19, 2008, under number 41597 on Nov. 19, 2008, and under number41599 on Nov. 19, 2008, respectively.

In another embodiment the targeted binding agent may comprise a sequencecomprising any one of the CDR1, CDR2 or CDR3 of the light chain variabledomain sequences encoded by a polynucleotide in a plasmid designated2.7A4_G, 2.14H9_G and 2.9D10_NG which were deposited under number 41598on Nov. 19, 2008, under number 41597 on Nov. 19, 2008, or under number41599 on Nov. 19, 2008, respectively.

In one embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designated2.7A4_G which was deposited at the NCIMB under deposit number 41598 onNov. 19, 2008 and a light chain variable domain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designated2.7A4_G which was deposited at the NCIMB under deposit number 41598 onNov. 19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designated 2.7A4_Gwhich was deposited at the NCIMB under deposit number 41598 on Nov. 19,2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a light chain variable domain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designated 2.7A4_Gwhich was deposited at the NCIMB under deposit number 41598 on Nov. 19,2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designated 2.7A4_Gwhich was deposited at the NCIMB under deposit number 41598 on Nov. 19,2008 and a light chain variable domain amino acid sequence comprising atleast one, at least two, or at least three of the CDRs of the antibodyencoded by the polynucleotide in plasmid designated 2.7A4_G which wasdeposited at the NCIMB under deposit number 41598 on Nov. 19, 2008.

In one embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designated2.14H9_G which was deposited at the NCIMB under number 41597 on Nov. 19,2008.

In one embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designated2.14H9_G which was deposited at the NCIMB under number 41597 on Nov. 19,2008, and a light chain variable domain amino acid sequence comprising aCDR3 encoded by the polynucleotide in plasmid designated 2.14H9_G whichwas deposited at the NCIMB under number 41597 on Nov. 19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designated2.14H9_G which was deposited at the NCIMB under number 41597 on Nov. 19,2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a light chain variable domain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designated2.14H9_G which was deposited at the NCIMB under number 41597 on Nov. 19,2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designated2.14H9_G which was deposited at the NCIMB under number 41597 on Nov. 19,2008 and a light chain variable domain amino acid sequence comprising atleast one, at least two, or at least three of the CDRs of the antibodyencoded by the polynucleotide in plasmid designated 2.14H9_G which wasdeposited at the NCIMB under number 41597 on Nov. 19, 2008.

In one embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designated2.9D10_NG which was deposited at the NCIMB under number 41599 on Nov.19, 2008.

In one embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designated2.9D10_NG which was deposited at the NCIMB under number 41599 on Nov.19, 2008 and a light chain variable domain amino acid sequencecomprising a CDR3 encoded by the polynucleotide in plasmid designated2.9D10_NG which was deposited at the NCIMB under number 41599 on Nov.19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designated2.9D10_NG which was deposited at the NCIMB under number 41599 on Nov.19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a light chain variable domain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designated2.9D10_NG which was deposited at the NCIMB under number 41599 on Nov.19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designated2.9D10_NG which was deposited at the NCIMB under number 41599 on Nov.19, 2008 and a light chain variable domain amino acid sequencecomprising at least one, at least two, or at least three of the CDRs ofthe antibody encoded by the polynucleotide in plasmid designated2.9D10_NG which was deposited at the NCIMB under number 41599 on Nov.19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable sequence of an antibodyencoded by the polynucleotide in plasmid designated 2.7A4_G which wasdeposited at the NCIMB number 41598 on Nov. 19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable sequence of an antibodyencoded by the polynucleotide in plasmid designated 2.14H9_G which wasdeposited at the NCIMB under number 41597 on Nov. 19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain sequence of anantibody encoded by the polynucleotide in plasmid designated 2.9D10_NGwhich was deposited at the NCIMB under number 41599 on Nov. 19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a light chain variable domain of an antibody encodedby the polynucleotide in plasmid designated 2.7A4_G which was depositedat the NCIMB under number 41598 on Nov. 19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a light chain variable domain of an antibody encodedby the polynucleotide in plasmid designated 2.14H9_G which was depositedat the NCIMB under number 41597 on Nov. 19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a light chain variable domain of an antibody encodedby the polynucleotide in plasmid designated 2.9D10_NG which wasdeposited at the NCIMB under number 41599 on Nov. 19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain sequence of anantibody encoded by the polynucleotide in plasmid designated 2.7A4_Gwhich was deposited at the NCIMB under number 41598 on Nov. 19, 2008 anda light chain variable domain sequence of an antibody encoded by thepolynucleotide in plasmid designated 2.7A4_G which was deposited at theNCIMB under number 41598 on Nov. 19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a light chain variable domain sequence of anantibody encoded by the polynucleotide in plasmid designated 2.14H9_Gwhich was deposited at the NCIMB under number 41597 on Nov. 19, 2008 anda heavy chain variable domain sequence of an antibody encoded by thepolynucleotide in plasmid designated 2.14H9_G which was deposited at theNCIMB under number 41597 on Nov. 19, 2008.

In another embodiment, a targeted binding agent or an antibody of theinvention comprises a heavy chain variable domain sequence of anantibody encoded by the polynucleotide in plasmid designated 2.9D10_NGwhich was deposited at the NCIMB under number 41599 on Nov. 19, 2008 anda light chain variable domain sequence of an antibody encoded by thepolynucleotide in plasmid designated 2.9D10_NG which was deposited atthe NCIMB under number 41599 on Nov. 19, 2008.

In one embodiment a targeted binding agent or an antibody may comprise asequence comprising a heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2)and heavy chain CDR3 (HCDR3) selected from any one of the sequencesshown in Table 8. In one embodiment a targeted binding agent or anantibody may comprise a sequence comprising a light chain CDR1 (LCDR1),light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) selected from anyone of the sequences shown in Table 9. In one embodiment a targetedbinding agent or an antibody may comprise a sequence comprising a HCDR1,HCDR2 and HCDR3 selected from any one of the CDRs of antibodies 2.9D10,2.7A4, 2.14H9, 3.15G8, 2.20A8, or 3.18G1. In one embodiment a targetedbinding agent or an antibody may comprise a sequence comprising a LCDR1,LCDR2 and LCDR3 selected from any one of the CDRs of antibodies 2.9D10,2.7A4, 2.14H9, 3.15G8, 2.20A8, or 3.18G1.

A further embodiment is a targeted binding agent or an antibody thatspecifically binds to B7-H1 and comprises a sequence comprising one ofthe CDR2 and one of the CDR3 sequences shown in Table 9. In a furtherembodiment the targeted binding agent or antibody further comprises asequence comprising: a CDR3 sequence as shown in Table 8. In a furtherembodiment the targeted binding agent or antibody further comprises asequence comprising: a CDR2 and a CDR3 sequence as shown in Table 8and/or Table 9. In a further embodiment the targeted binding agent orantibody further comprises a sequence comprising: a CDR1, a CDR2 and aCDR3 sequence as shown in Table 8 and/or Table 9.

In another embodiment the targeted binding agent or antibody maycomprise a sequence comprising any one of a CDR1, a CDR2 or a CDR3 ofany one of the fully human monoclonal antibodies 2.7A4, 2.14H9 or2.9D10, as shown in Table 8. In another embodiment the targeted bindingagent or antibody may comprise a sequence comprising any one of a CDR1,a CDR2 or a CDR3 of any one of the fully human monoclonal antibodies2.7A4, 2.14H9 or 2.9D10, as shown in Table 9. In one embodiment thetargeted binding agent or antibody may comprise a sequence comprising aCDR1, a CDR2 and a CDR3 of any one of fully human monoclonal antibodies2.7A4, 2.14H9, 2.9D10, 2.7A4OPT or 2.14H9OPT, as shown in Table 8. Inanother embodiment the targeted binding agent or antibody may comprise asequence comprising a CDR1, a CDR2 and a CDR3 of any one of fully humanmonoclonal antibodies 2.7A4, 2.14H9, 2.9D10, 2.7A4OPT or 2.14H9OPT, asshown in Table 9.

In another embodiment the targeted binding agent or antibody comprises asequence comprising the CDR1, CDR2 and CDR3 sequence of fully humanmonoclonal antibody 2.7A4 as shown in Table 8 and the CDR1, CDR2 andCDR3 sequence of fully human monoclonal antibody 2.7A4 as shown in Table9. In another embodiment the targeted binding agent or antibodycomprises a sequence comprising the CDR1, CDR2 and CDR3 sequence offully human monoclonal antibody 2.14H9 as shown in Table 8 and the CDR1,CDR2 and CDR3 sequence of fully human monoclonal antibody 2.14H9 asshown in Table 9. In another embodiment the targeted binding agent orantibody comprises a sequence comprising the CDR1, CDR2 and CDR3sequence of fully human monoclonal antibody 2.9D10 as shown in Table 8and the CDR1, CDR2 and CDR3 sequence of fully human monoclonal antibody2.9D10 as shown in Table 9. In some embodiments, the antibody is a fullyhuman monoclonal antibody.

It is noted that those of ordinary skill in the art can readilyaccomplish CDR determinations. See for example, Kabat et al., Sequencesof Proteins of Immunological Interest, Fifth Edition, NIH Publication91-3242, Bethesda Md. (1991), vols. 1-3. Kabat provides multiplesequence alignments of immunoglobulin chains from numerous speciesantibody isotypes. The aligned sequences are numbered according to asingle numbering system, the Kabat numbering system. The Kabat sequenceshave been updated since the 1991 publication and are available as anelectronic sequence database (presently available from the KabatDatabase Website; see also Nucleic Acids Research, 2000, 28(1),214-218). Any immunoglobulin sequence can be numbered according to Kabatby performing an alignment with the Kabat reference sequence.

Accordingly, the Kabat numbering system provides a uniform system fornumbering immunoglobulin chains.

A further embodiment of the invention is a targeted binding agent orantibody comprising a sequence comprising the contiguous sequencespanning the framework regions and CDRs, specifically from FR1 throughFR4 or CDR1 through CDR3, of any one of the sequences 2.9D10, 2.7A4,2.14H9, 3.15G8, 2.20A8, 3.18G1, 2.7A4OPT, or 2.14H9OPT, or as shown inTable 8 or Table 9. A further embodiment of the invention is a targetedbinding agent or antibody comprising a sequence comprising thecontiguous sequence spanning the framework regions and CDRs,specifically from FR1 through FR4 or CDR1 through CDR3, of any one ofthe sequences 2.9D10, 2.7A4, 2.14H9, 3.15G8, 2.20A8, 3.18G1, 2.7A4OPT,or 2.14H9OPT or as shown in Table 8 and Table 9. In one embodiment thetargeted binding agent or antibody comprises a sequence comprising thecontiguous sequences spanning the framework regions and CDRs,specifically from FR1 through FR4 or CDR1 through CDR3, of any one ofthe sequences of monoclonal antibodies 2.9D10, 2.7A4, 2.14H9, 3.15G8,2.20A8, 3.18G1, 2.7A4OPT, or 2.14H9OPT or as shown in Table 8 or Table9. A further embodiment of the invention is a targeted binding agent orantibody comprising a sequence comprising the contiguous sequencespanning the framework regions and CDRs, specifically from FR1 throughFR4 or CDR1 through CDR3, of any one of the sequences of monoclonalantibodies 2.9D10, 2.7A4, 2.14H9, 3.15G8, 2.20A8, 3.18G1, 2.7A4OPT, or2.14H9OPT or as shown in Table 8 and Table 9. In some embodiments, theantibody is a fully human monoclonal antibody.

In another embodiment, a targeted binding agent or antibody of theinvention comprises a CDR3 sequence as shown in Table 8 or 9; or any oneof a CDR1, a CDR2 or a CDR3 sequence as shown in Table 8 or 9; or aCDR1, a CDR2 and a CDR3 sequence of a light chain variable domainsequence as shown in Table 8; or a CDR1, a CDR2 and a CDR3 sequence of aheavy chain variable domain sequence as shown as shown in Table 9.

One embodiment provides a targeted binding agent or antibody, orantigen-binding portion thereof, wherein the agent or antibody, orantigen-binding portion thereof, comprises a sequence comprising SEQ IDNO.:2, SEQ ID NO.:7, SEQ ID NO.:12, SEQ ID NO.:17, SEQ ID NO.:22, SEQ IDNO.:27, SEQ ID NO.:32, SEQ ID NO.:37, SEQ ID NO.:42, SEQ ID NO.:47, SEQID NO.:52, SEQ ID NO.:57, SEQ ID NO.:62, SEQ ID NO.:67, SEQ ID NO.:72,or SEQ ID NO.:77.

One embodiment provides a targeted binding agent or antibody, orantigen-binding portion thereof, wherein the agent or antibody, orantigen-binding portion thereof, comprises a heavy chain sequencecomprising the sequence of SEQ ID NO.:2. In one embodiment, the targetedbinding agent or antibody, or antigen-binding portion thereof, furthercomprises a light chain sequence comprising the sequence of SEQ IDNO.:7. In some embodiments, the antibody is a fully human monoclonalantibody.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain variable domainhaving at least 90% identity to the amino acid of SEQ ID NO:2 andcomprises a light chain variable domain having at least 90% identity tothe amino acid sequence of SEQ ID NO:7.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain sequencecomprising the sequence of SEQ ID NO.:12. In one embodiment, thetargeted binding agent or antibody, or antigen-binding portion thereof,further comprises a light chain sequence comprising the sequence of SEQID NO.:17. In some embodiments, the antibody is a fully human monoclonalantibody.

In one embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain variable domainhaving at least 90% identity to the amino acid of SEQ ID NO:12 andcomprises a light chain variable domain having at least 90% identity tothe amino acid sequence of SEQ ID NO:17.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain sequencecomprising the sequence of SEQ ID NO.:22. In another embodiment, thetargeted binding agent or antibody, or antigen-binding portion thereof,further comprises a light chain sequence comprising the sequence of SEQID NO.:27. In some embodiments, the antibody is a fully human monoclonalantibody.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain variable domainhaving at least 90% identity to the amino acid of SEQ ID NO:22 andcomprises a light chain variable domain having at least 90% identity tothe amino acid sequence of SEQ ID NO:27.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain sequencecomprising the sequence of SEQ ID NO.:32. In another embodiment, thetargeted binding agent or antibody, or antigen-binding portion thereof,further comprises a light chain sequence comprising the sequence of SEQID NO.:37. In some embodiments, the antibody is a fully human monoclonalantibody.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain variable domainhaving at least 90% identity to the amino acid of SEQ ID NO:32 andcomprises a light chain variable domain having at least 90% identity tothe amino acid sequence of SEQ ID NO:37.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain sequencecomprising the sequence of SEQ ID NO.:42. In another embodiment, thetargeted binding agent or antibody, or antigen-binding portion thereof,further comprises a light chain sequence comprising the sequence of SEQID NO.:47. In some embodiments, the antibody is a fully human monoclonalantibody.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain variable domainhaving at least 90% identity to the amino acid of SEQ ID NO:42 andcomprises a light chain variable domain having at least 90% identity tothe amino acid sequence of SEQ ID NO:47.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain sequencecomprising the sequence of SEQ ID NO.:52. In another embodiment, thetargeted binding agent or antibody, or antigen-binding portion thereof,further comprises a light chain sequence comprising the sequence of SEQID NO.:57. In some embodiments, the antibody is a fully human monoclonalantibody.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain variable domainhaving at least 90% identity to the amino acid of SEQ ID NO:52 andcomprises a light chain variable domain having at least 90% identity tothe amino acid sequence of SEQ ID NO:57.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain sequencecomprising the sequence of SEQ ID NO.:62. In another embodiment, thetargeted binding agent or antibody, or antigen-binding portion thereof,further comprises a light chain sequence comprising the sequence of SEQID NO.:67. In some embodiments, the antibody is a fully human monoclonalantibody.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain variable domainhaving at least 90% identity to the amino acid of SEQ ID NO:62 andcomprises a light chain variable domain having at least 90% identity tothe amino acid sequence of SEQ ID NO:67.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain sequencecomprising the sequence of SEQ ID NO.:72. In another embodiment, thetargeted binding agent or antibody, or antigen-binding portion thereof,further comprises a light chain sequence comprising the sequence of SEQID NO.:77. In some embodiments, the antibody is a fully human monoclonalantibody.

In another embodiment the targeted binding agent or antibody, orantigen-binding portion thereof, comprises a heavy chain variable domainhaving at least 90% identity to the amino acid of SEQ ID NO:72 andcomprises a light chain variable domain having at least 90% identity tothe amino acid sequence of SEQ ID NO:77.

In one embodiment, the targeted binding agent or antibody comprisesvariants or derivatives of the CDRs disclosed herein, the contiguoussequences spanning the framework regions and CDRs (specifically from FR1through FR4 or CDR1 through CDR3), the light or heavy chain sequencesdisclosed herein, or the antibodies disclosed herein. Variants includetargeted binding agents or antibodies comprising sequences which have asmany as twenty, sixteen, ten, nine or fewer, e.g. one, two, three, four,five or six amino acid additions, substitutions, deletions, and/orinsertions in any of the CDR1, CDR2 or CDR3s as shown in Table 8 orTable 9, the contiguous sequences spanning the framework regions andCDRs (specifically from FR1 through FR4 or CDR1 through CDR3) as shownin Table 8 or Table 9, the light or heavy chain sequences disclosedherein, or with the monoclonal antibodies disclosed herein. Variantsinclude targeted binding agents or antibodies comprising sequences whichhave one, two or three, amino acid additions, substitutions, deletions,and/or insertions in any of the CDR1, CDR2 or CDR3s as shown in Table 8or Table 9, the contiguous sequences spanning the framework regions andCDRs (specifically from FR1 through FR4 or CDR1 through CDR3) as shownin Table 8 or Table 9, the light or heavy chain sequences disclosedherein, or with the monoclonal antibodies disclosed herein. Variantsinclude targeted binding agents or antibodies comprising sequences whichhave at least about 60, 70, 80, 85, 90, 95, 98 or about 99% amino acidsequence identity with any of the CDR1, CDR2 or CDR3s as shown in Table8 or Table 9, the contiguous sequences spanning the framework regionsand CDRs (specifically from FR1 through FR4 or CDR1 through CDR3) asshown in Table 8 or Table 9, the light or heavy chain sequencesdisclosed herein, or with the monoclonal antibodies disclosed herein.The percent identity of two amino acid sequences can be determined byany method known to one skilled in the art, including, but not limitedto, pairwise protein alignment. In one embodiment variants comprisechanges in the CDR sequences or light or heavy chain sequences disclosedherein that are naturally occurring or are introduced by in vitroengineering of native sequences using recombinant DNA techniques ormutagenesis techniques. Naturally occurring variants include those whichare generated in vivo in the corresponding germline nucleotide sequencesduring the generation of an antibody to a foreign antigen.

In one embodiment variants include targeted binding agents or antibodiescomprising sequences which have (a) a VH CDR1 having an amino acidsequence identical to or comprising 1, 2, or 3 amino acid residuesubstitutions relative to SEQ ID NO: 3;

(b) a VH CDR2 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 4;

(c) a VH CDR3 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 5;

(d) a VL CDR1 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to VL CDR1 of SEQID NO: 8;

(e) a VL CDR2 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 9;and

(f) a VL CDR3 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 10.

In another embodiment, variants include targeted binding agents orantibodies comprising sequences which have (a) a VH CDR1 having an aminoacid sequence identical to or comprising 1, 2, or 3 amino acid residuesubstitutions relative to SEQ ID NO: 23;

(b) a VH CDR2 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 24;

(c) a VH CDR3 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 25;

(d) a VL CDR1 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to VL CDR1 of SEQID NO: 28;

(e) a VL CDR2 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 29;and

(f) a VL CDR3 having an amino acid sequence identical to or comprising1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 30.

In one embodiment the derivative may be a heteroantibody, that is anantibody in which two or more antibodies are linked together.Derivatives include antibodies which have been chemically modified.Examples include covalent attachment of one or more polymers, such aswater-soluble polymers, N-linked, or O-linked carbohydrates, sugars,phosphates, and/or other such molecules. The derivatives are modified ina manner that is different from naturally occurring or startingantibody, either in the type or location of the molecules attached.Derivatives further include deletion of one or more chemical groupswhich are naturally present on the antibody.

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 2. In someembodiments of the invention, the targeted binding agent or antibodycomprises a sequence comprising SEQ ID NO.: 2, wherein SEQ ID NO.: 2comprises any one of the unique combinations of germline andnon-germline residues indicated by each row of Table 10. In someembodiments of the invention, the targeted binding agent or antibodycomprises a sequence comprising SEQ ID NO.: 2, wherein SEQ ID NO.: 2comprises any one, any two, any three, any four or all five of thegermline residues as indicated in Table 10. In some embodiments of theinvention, the targeted binding agent or antibody comprises a sequencecomprising SEQ ID NO.: 2, wherein SEQ ID NO.: 2 comprises any one of theunique combinations of germline and non-germline residues indicated byeach row of Table 10. In some embodiments of the invention, the targetedbinding agent or antibody comprises a sequence comprising SEQ ID NO.: 2,wherein SEQ ID NO.: 2 comprises any one, any two, any three, any four,any five, or all five of the germline residues as indicated in Table 10.

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 7, wherein SEQ IDNO.: 7 comprises any one of the unique combinations of germline andnon-germline residues indicated by each row of Table 11 and any one ofthe unique combinations of germline and non-germline residues indicatedby each row of Table 11. In some embodiments of the invention, thetargeted binding agent or antibody comprises a sequence comprising SEQID NO.: 7, wherein SEQ ID NO.: 7 comprises any one, any two, any three,any four, any five, for all five of the germline residues as indicatedin Table 11.

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 12. In someembodiments of the invention, the targeted binding agent or antibodycomprises a sequence comprising SEQ ID NO.: 12, wherein SEQ ID NO.: 12comprises any one of the unique combinations of germline andnon-germline residues indicated by each row of Table 12. In someembodiments of the invention, the targeted binding agent or antibodycomprises a sequence comprising SEQ ID NO.: 12, wherein SEQ ID NO.: 12comprises any one, any two or all two of the germline residues asindicated in Table 12.

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 17. In someembodiments of the invention, the targeted binding agent or antibodycomprises a sequence comprising SEQ ID NO.: 17, wherein SEQ ID NO.: 17comprises any one of the unique combinations of germline andnon-germline residues indicated by each row of Table 13. In someembodiments of the invention, the targeted binding agent or antibodycomprises a sequence comprising SEQ ID NO.: 17, wherein SEQ ID NO.: 17comprises any one, any two, any three, any four or all four of thegermline residues as indicated in Table 13.

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 27. In someembodiments of the invention, the targeted binding agent or antibodycomprises a sequence comprising SEQ ID NO.: 27, wherein SEQ ID NO.: 27comprises any one of the unique combinations of germline andnon-germline residues indicated by each row of Table 14. In someembodiments of the invention, the targeted binding agent or antibodycomprises a sequence comprising SEQ ID NO.: 27, wherein SEQ ID NO.: 27comprises any one, any two, any three or all three of the germlineresidues as indicated in Table 14.

A further embodiment of the invention is a targeted binding agent orantibody which competes for binding to B7-H1 with the targeted bindingagent or antibodies of the invention. In another embodiment of theinvention there is an antibody which competes for binding to B7-H1 withthe targeted binding agent or antibodies of the invention. In anotherembodiment the targeted binding agent or antibody competes for bindingto B7-H1 with any one of fully human monoclonal antibodies 2.7A4, 2.14H9or 2.9D10, 2.7A4OPT or 2.14H9OPT. “Competes” indicates that the targetedbinding agent or antibody competes for binding to B7-H1 with any one offully human monoclonal antibodies 2.7A4, 2.14H9 or 2.9D10, 2.7A4OPT or2.14H9OPT, i.e. competition is unidirectional.

Embodiments of the invention include a targeted binding agent orantibody which cross competes with any one of fully human monoclonalantibodies 2.7A4, 2.14H9 or 2.9D10, 2.7A4OPT or 2.14H9OPT for binding toB7-H1. “Cross competes” indicates that the targeted binding agent orantibody competes for binding to B7-H1 with any one of fully humanmonoclonal antibodies 2.7A4, 2.14H9 or 2.9D10, 2.7A4OPT or 2.14H9OPT,and vice versa, i.e. competition is bidirectional.

A further embodiment of the invention is a targeted binding agent orantibody that binds to the same epitope or epitopes on the extracellulardomain of human B7-H1 as the targeted binding agent or antibodies of theinvention. Embodiments of the invention also include a targeted bindingagent or antibody that binds to the same epitope or epitopes on theextracellular domain of human B7-H1 as any one of fully human monoclonalantibodies 2.7A4, 2.14H9 or 2.9D10, 2.7A4OPT or 2.14H9OPT.

In one embodiment, the targeted binding agent or antibody binds anepitope on human B7-H1 including at least one or more of the followingamino acids selected from the group consisting of Asp at position 122and Arg at position 125. In another embodiment, an antibody of theinvention binds an epitope on human B7-H1 comprising at least two of thefollowing three amino acid residues of Asp at position 122, Arg atposition 125 and Arg at position 113. In another embodiment, theantibody binds an epitope on human B7-H1, wherein the antibody exhibitsno binding to Ile at position 54, Ser at position 117 and Ala atposition 121 on human B7-H1. In yet a further embodiment, an antibody ofthe invention loses its ability to bind to human B7-H1 if the Arg atposition 113 is mutated to an Ala, or to a Tyr, or to a Leu asdetermined by a competition assay as compared to binding to wild-typeB7-H1. In yet a further embodiment, an antibody of the invention losesits ability to bind to human B7-H1 if the Arg at position 125 is mutatedto an Ala, or to a Gin, or to a Ser as determined by a competition assayas compared to binding to wild-type B7-H1. In yet a further embodiment,an antibody of the invention retains its ability to bind to human B7-H1if the Arg at position 123 is mutated to an Ala, or to a Phe, or to aThr as determined by a competition assay as compared to binding towild-type B7-H1. In this example, the antibody is 2.14H9. In anotherexample, the antibody is 2.14H9OPT.

In one embodiment, the targeted binding agent or antibody binds anepitope on the extracellular domain of human B7-H1 comprising at leastone or more of the following amino acids Asp at position 122 and Thr atposition 20. In one embodiment, the antibody binds at least two of thefollowing three amino acid residues of Phe at position 19, Thr atposition 20 and Asp at position 122 on human B7-H1. In anotherembodiment, the shows no binding to at least one of the following threeamino acid residues of Ile at position 54, Met at position 115, Ser atposition 117 and Ala at position 121 on human B7-H1. In yet a furtherembodiment, an antibody of the invention loses its ability to bind tohuman B7-H1 if the Phe at position 19 is mutated to an Ala, or to a Gly,or to a Ser as determined by a competition assay as compared to bindingto wild-type B7-H1. In yet a further embodiment, an antibody of theinvention loses its ability to bind to human B7-H1 if the Thr atposition 20 is mutated to an Ala, or to a Val, or to a Asp as determinedby a competition assay as compared to binding to wild-type B7-H1. In yeta further embodiment, an antibody of the invention loses its ability tobind to human B7-H1 if the Asp at position 122 is mutated to an Asn, orto a Glu as determined by a competition assay as compared to binding towild-type B7-H1. In yet a further embodiment, an antibody of theinvention retains its ability to bind to human B7-H1 if the Arg atposition 123 is mutated to an Ala, or to a Phe, or to a Thr asdetermined by a competition assay as compared to binding to wild-typeB7-H1. In one example, the antibody is 2.7A4. In another example, theantibody is 2.7A4OPT.

In one embodiment, the targeted binding agent is a bispecific antibody.A bispecific antibody is an antibody that has binding specificity for atleast two different epitopes on the same or on different proteins.Methods for making bispecific antibodies are known in the art. (See, forexample, Millstein et al., Nature, 305:537-539 (1983); Traunecker etal., IMBO J., 10:3655-3659 (1991); Suresh et al., Methods in Enzymology,121:210 (1986); Kostelny et al., J. Immunol., 148(5):1547-1553 (1992);Hollinger et al., Proc. Natl Acad. Sci. USA, 90:6444-6448 (1993); Gruberet a., J. Immunol., 152:5368 (1994); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,81: 95,731,168; 4,676,980; and4,676,980, WO 94/04690; WO 91/00360; WO 92/200373; WO 93/17715; WO92/08802; and EP 03089.)

Embodiments of the invention described herein relate to monoclonalantibodies that specifically bind B7-H1 and affect B7-H1 function. Otherembodiments relate to fully human antibodies that specifically bindB7-H1 and preparations thereof with desirable properties from atherapeutic perspective, including high binding affinity for B7-H1, highselectivity for inhibition of B7-H1 signaling, low toxicity, the abilityto block PD-1 receptor activity, the ability to inhibit B7-H1-inducedtumour cell survival through immune suppression, the ability to inhibitB7-H1 mediated repression of anti-tumour immunity, which may in turninhibit proliferation or invasion-related diseases include neoplasticdiseases, and/or the ability of tumour cells to grow in vitro and invivo. Still other embodiments relate to a method of repressingB7-H1-mediated T cell inhibition in an animal by administering to ananimal in need thereof an effective amount of a composition comprisingthe antibodies of the invention. Still other embodiments relate to fullyhuman antibodies that specifically bind B7-H1 and preparations thereofthat do not result in a significant Human Anti-Chimeric Antibody (HACA)response, thereby allowing for repeated administration.

In one embodiment of the invention there is provided nucleic acidmolecule encoding any of the targeted binding agents or antibodies ofthe invention. In one embodiment is a nucleic acid molecule encoding thelight chain or the heavy chain of an antibody of the invention. In oneembodiment, the nucleic acid molecule encodes the light chain or theheavy chain of a fully human monoclonal antibody of any of theantibodies described herein. In one embodiment, the nucleic acidmolecule encodes the light chain or the heavy chain of any one of thefully human monoclonal antibodies 2.7A4, 2.14H9, 2.9D10, 2.7A4OPT and2.14H9OPT. In another embodiment, the nucleic acid molecule encodes thelight chain and the heavy chain of any one of the fully human monoclonalantibodies 2.7A4, 2.14H9, 2.9D10, 2.7A4OPT and 2.14H9OPT. The inventionalso encompasses polynucleotides that hybridize under stringent or lowerstringency hybridization conditions, as defined herein, topolynucleotides that encode any of the targeted binding agents orantibodies described herein.

In another embodiment of the invention there is provided a vectorcomprising a nucleic acid molecule or molecules as describedhereinabove, wherein the vector encodes a targeted binding agent asdescribed hereinabove. In one embodiment of the invention there isprovided a vector comprising a nucleic acid molecule or molecules asdescribed hereinabove, wherein the vector encodes a light chain and/or aheavy chain of an antibody as defined hereinabove. In one embodiment,the vector comprises a nucleic acid molecule encoding the light chainand/or the heavy chain of a fully human monoclonal antibody. In oneembodiment, the vector comprises a nucleic acid molecule encoding thelight chain or the heavy chain of any one of the fully human monoclonalantibodies 2.7A4, 2.14H9, 2.9D10, 2.7A4OPT and 2.14H9OPT. In anotherembodiment, the vector comprises a nucleic acid molecule encoding thelight chain and the heavy chain of any one of the fully human monoclonalantibodies 2.7A4, 2.14H9, 2.9D10, 2.7A4OPT and 2.14H9OPT.

In a further embodiment there is provided a host cell transformed withany of the nucleic acid molecules as described hereinabove. In anotherembodiment of the invention there is provided a host cell comprising thevector comprising the nucleic acid molecule as described hereinabove. Inone embodiment the host cell may comprise more than one vector.

As known in the art, antibodies can advantageously be, for example,polyclonal, oligoclonal, monoclonal, chimeric, humanised, and/or fullyhuman antibodies.

It will be appreciated that embodiments of the invention are not limitedto any particular form of an antibody or method of generation orproduction. In some embodiments of the invention, the targeted bindingagent is a binding fragment of a fully human monoclonal antibody. Forexample, the targeted binding agent can be a full-length antibody (e.g.,having an intact human Fc region) or an antibody binding fragment (e.g.,a Fab, Fab′ or F(ab′)₂, Fv, dAb or other well known antibody fragment,as described in more detail below). In addition, the antibodies can besingle-domain antibodies such as camelid or human single VH or VLdomains that bind to B7-H1, such as a dAb fragment.

Embodiments of the invention described herein also provide cells forproducing these antibodies. Examples of cells include hybridomas, orrecombinantly created cells, such as Chinese hamster ovary (CHO) cells,variants of CHO cells (for example DG44) and NSO cells that produceantibodies against B7-H1. Additional information about variants of CHOcells can be found in Andersen and Reilly (2004) Current Opinion inBiotechnology 15, 456-462 which is incorporated herein in its entiretyby reference. The antibody can be manufactured from a hybridoma thatsecretes the antibody, or from a recombinantly engineered cell that hasbeen transformed or transfected with a gene or genes encoding theantibody.

In addition, one embodiment of the invention is a method of producing atargeted binding agent or an antibody of the invention by culturing hostcells under conditions wherein a nucleic acid molecule is expressed toproduce the targeted binding agent or antibody followed by recovery ofthe targeted binding agent or antibody. In one embodiment is a method ofproducing an antibody of the invention by culturing host cells underconditions wherein a nucleic acid molecule is expressed to produce theantibody, followed by recovery of the antibody. Still other embodimentsinclude an antibody of the invention produced by the method of culturinga host cell which expresses an antibody encoded by a nucleic acidmolecule encoding an antibody of the invention, and isolating saidantibody from said culture.

It should be realised that embodiments of the invention also include anynucleic acid molecule which encodes an antibody or fragment of anantibody of the invention including nucleic acid sequences optimised forincreasing yields of antibodies or fragments thereof when transfectedinto host cells for antibody production.

A further embodiment herein includes a method of producing antibodiesthat specifically bind to B7-H1 and inhibit the biological activity ofB7-H1, by immunising a mammal with cells expressing B7-H1, isolated cellmembranes containing B7-H1, purified B7-H1, or a fragment thereof,and/or one or more orthologous sequences or fragments thereof. A furtherembodiment herein includes a method of producing high affinityantibodies that specifically bind to B7-H1 and inhibit the biologicalactivity of B7-H1, by immunising a mammal with cells expressing B7-H1,isolated cell membranes containing B7-H1, purified B7-H1, or a fragmentthereof, and/or one or more orthologous sequences or fragments thereof.

Other embodiments are based upon the generation and identification ofisolated antibodies that bind specifically to B7-H1 and inhibit thebiological activity of B7-H1. B7-H1 is expressed on a number of tumourtypes. Antibodies that specifically bind to B7-H1 can preventB7-H1-mediated tumour cell survival and inhibit B7-H1 mediatedrepression of anti-tumour immune responses through immune suppression,this can in turn reduce tumour cell invasion, metastasis, tumour growth,and other properties.

In addition, the antibody can be manufactured from a hybridoma thatsecretes the antibody, or from a recombinantly engineered cell that hasbeen transformed or transfected with a gene or genes encoding theantibody.

In one embodiment there is a hybridoma that produces the targetedbinding agent or antibody of the invention. In one embodiment there is ahybridoma that produces the light chain and/or the heavy chain of anantibody of the invention. In one embodiment the hybridoma may produce alight chain and/or a heavy chain of a fully human monoclonal antibody.In another embodiment, the hybridoma produces the light chain and/or theheavy chain of the fully human monoclonal antibody 2.7A4, 2.14H9,2.9D10, 2.7A4OPT and 2.14H9OPT. Alternatively the hybridoma may producean antibody that binds to the same epitope or epitopes as fully humanmonoclonal antibody 2.7A4, 2.14H9, 2.9D10, 2.7A4OPT and 2.14H9OPT.Alternatively the hybridoma may produce an antibody that competes forbinding to B7-H1 with fully human monoclonal antibody 2.7A4, 2.14H9,2.9D10, 2.7A4OPT and 2.14H9OPT. Alternatively the hybridoma may producean antibody that cross-competes for binding to B7-H1 with fully humanmonoclonal antibody 2.7A4, 2.14H9, 2.9D10, 2.7A4OPT and 2.14H9OPT.

In other embodiments the invention provides compositions, including atargeted binding agent or antibody of the invention or binding fragmentthereof, and a pharmaceutically acceptable carrier.

Still further embodiments of the invention include methods of treating aproliferative or invasion-related disease in an animal by administeringto the animal a therapeutically effective dose of a targeted bindingagent of the invention. In certain embodiments the method furthercomprises selecting an animal in need of treatment for a proliferativeor invasion-related disease, and administering to the animal atherapeutically effective dose of a targeted binding agent of theinvention. In certain embodiments, the animal is human. In certainembodiments, the targeted binding agent is a fully human monoclonalantibody. In certain embodiments, the targeted binding agent is anantibody of the invention and may be selected from the group consistingof 2.7A4, 2.14H9 or 2.9D10, 2.7A4OPT or 2.14H9OPT.

Still further embodiments of the invention include methods of inhibitingcell proliferation or invasion-related disease, with a B7-H1 mediatedcomponent, in an animal by administering to the animal a therapeuticallyeffective dose of a targeted binding agent of the invention. In certainembodiments the method further comprises selecting an animal in need oftreatment for proliferation or invasion-related disease, with a B7-H1mediated component, and administering to said animal a therapeuticallyeffective dose of a targeted binding agent of the invention. In certainembodiments, the animal is human. In certain embodiments, the targetedbinding agent is a fully human monoclonal antibody. In certainembodiments, the targeted binding agent is an antibody of the inventionand may be selected from the group consisting of 2.7A4, 2.14H9 or2.9D10, 2.7A4OPT or 2.14H9OPT.

Still further embodiments of the invention include methods of inhibitingtumour cell invasion, cellular metastasis or tumour growth in an animalby administering to the animal a therapeutically effective dose of atargeted binding agent of the invention. In certain embodiments themethod further comprises selecting an animal in need of treatment fortumour cell, invasion, cellular metastasis or tumour growth, andadministering to the animal a therapeutically effective dose of atargeted binding agent of the invention. In certain embodiments, theanimal is human. In certain embodiments, the targeted binding agent is afully human monoclonal antibody. In certain embodiments, the targetedbinding agent is an antibody of the invention and may be selected fromthe group consisting of 2.7A4, 2.14H9 or 2.9D10, 2.7A4OPT or 2.14H9OPT.

Still further embodiments of the invention include methods of treatingan animal suffering from a neoplastic disease by administering to theanimal a therapeutically effective dose of a targeted binding agent ofthe invention. In certain embodiments the method further comprisesselecting an animal in need of treatment for a neoplastic disease, andadministering to the animal a therapeutically effective dose of atargeted binding agent of the invention.

Still further embodiments of the invention include methods of treatingan animal suffering from a non-neoplastic disease by administering tothe animal a therapeutically effective dose of a targeted binding agentof the invention. In certain embodiments the method further comprisesselecting an animal in need of treatment for a non-neoplastic disease,and administering to the animal a therapeutically effective dose of atargeted binding agent of the invention.

Still further embodiments of the invention include methods of treatingan animal suffering from chronic viral infection by administering to theanimal a therapeutically effective dose of a targeted binding agent ofthe invention. In certain embodiments the method further comprisesselecting an animal in need of treatment for chronic viral infection,and administering to the animal a therapeutically effective dose of atargeted binding agent of the invention.

Still further embodiments of the invention include methods of treatingan animal suffering from a malignant tumour by administering to theanimal a therapeutically effective dose of a targeted binding agent ofthe invention. In certain embodiments the method further comprisesselecting an animal in need of treatment for a malignant tumour, andadministering to the animal a therapeutically effective dose of atargeted binding agent of the invention.

Still further embodiments of the invention include methods of treatingan animal suffering from a disease or condition associated with B7-H1expression by administering to the animal a therapeutically effectivedose of a targeted binding agent of the invention. In certainembodiments the method further comprises selecting an animal in need oftreatment for a disease or condition associated with B7-H1 expression,and administering to the animal a therapeutically effective dose of atargeted binding agent of the invention.

A malignant tumour may be selected from the group consisting of: solidtumours such as melanoma, skin cancers, small cell lung cancer,non-small cell lung cancer, glioma, hepatocellular (liver) carcinoma,gallbladder cancer, thyroid tumour, bone cancer, gastric (stomach)cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer,uterine cancer, vulval cancer, endometrial cancer, testicular cancer,bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidneycancer, renal cell carcinoma, colon cancer, colorectal, pancreaticcancer, esophageal carcinoma, brain/CNS cancers, head and neck cancers,neuronal cancers, mesothelioma, sarcomas, biliary (cholangiocarcinoma),small bowel adenocarcinoma, pediatric malignancies, epidermoidcarcinoma, sarcomas, cancer of the pleural/peritoneal membranes andleukaemia, including acute myeloid leukaemia, acute lymphoblasticleukaemia, and multiple myeloma.

Treatable proliferative or invasion-related diseases include neoplasticdiseases, such as, melanoma, skin cancer, small cell lung cancer,non-small cell lung cancer, salivary gland, glioma, hepatocellular(liver) carcinoma, gallbladder cancer, thyroid tumour, bone cancer,gastric (stomach) cancer, prostate cancer, breast cancer, ovariancancer, cervical cancer, uterine cancer, vulval cancer, endometrialcancer, testicular cancer, bladder cancer, lung cancer, glioblastoma,thyroid cancer, endometrial cancer, kidney cancer, colon cancer,colorectal cancer, pancreatic cancer, esophageal carcinoma, brain/CNScancers, neuronal cancers, head and neck cancers, mesothelioma,sarcomas, biliary (cholangiocarcinoma), small bowel adenocarcinoma,pediatric malignancies, epidermoid carcinoma, sarcomas, cancer of thepleural/peritoneal membranes and leukaemia, including acute myeloidleukaemia, acute lymphoblastic leukaemia, and multiple myeloma.Treatable chronic viral infections include HIV, hepatitis B virus (HBV),and hepatitis C virus (HCV) in humans, simian immunodeficiency virus(SIV) in monkeys, and lymphocytic choriomeningitis virus (LCMV) in mice.

Disease-related cell invasion and/or proliferation may be any abnormal,undesirable or pathological cell invasion and/or proliferation, forexample tumour-related cell invasion and/or proliferation.

In one embodiment, the neoplastic disease is a solid tumour selectedfrom any one of the following carcinomas of the breast, colon,colorectal, prostate, stomach, gastric, ovary, esophagus, pancreas,gallbladder, non-small cell lung cancer, thyroid, endometrium, head andneck, renal, renal cell carcinoma, bladder and gliomas.

In one embodiment the present invention is suitable for use ininhibiting B7-H1, in patients with a tumour which is dependent alone, orin part, on B7-H1.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation of amedicament for the treatment of an animal suffering from a proliferativeor invasion-related disease. In certain embodiments the use furthercomprises selecting an animal in need of treatment for a proliferativeor invasion-related disease.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation ofmedicament for the treatment of proliferation or invasion-relateddisease, with a B7-H1 mediated component, in an animal. In certainembodiments the use further comprises selecting an animal in need oftreatment for proliferation or invasion-related disease, with a B7-H1mediated component.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation ofmedicament for the treatment of tumour cell invasion, cellularmetastasis or tumour growth in an animal. In certain embodiments the usefurther comprises selecting an animal in need of treatment for tumourcell invasion, cellular metastasis or tumour.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation of amedicament for the treatment of an animal suffering from a neoplasticdisease. In certain embodiments the use further comprises selecting ananimal in need of treatment for a neoplastic disease.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation of amedicament for the treatment of an animal suffering from a disease wherethe etiology is associated with an infectious agent, such as, forexample, hepatocellular cancer, gastric cancer, or cervical cancer. Incertain embodiments the use further comprises selecting an animal inneed of treatment for a neoplastic disease.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation of amedicament for the treatment of an animal suffering from anon-neoplastic disease. In certain embodiments the use further comprisesselecting an animal in need of treatment for a non-neoplastic disease.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation of amedicament for the treatment of an animal suffering from chronic viralinfection. In certain embodiments the use further comprises selecting ananimal in need of treatment for a non-neoplastic disease. In still otherembodiments, the use further comprises ocular disease, inflammatorydisease, cardiovascular disease and sepsis.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation of amedicament for the treatment of an animal suffering from a malignanttumour. In certain embodiments the use further comprises selecting ananimal in need of treatment for a malignant tumour.

Still further embodiments of the invention include use of a targetedbinding agent or antibody of the invention in the preparation of amedicament for the treatment of an animal suffering from a disease orcondition associated with B7-H1 expression. In certain embodiments theuse further comprises selecting an animal in need of treatment for adisease or condition associated with B7-H1 expression.

Still further embodiments of the invention include a targeted bindingagent or antibody of the invention for use as a medicament for thetreatment of an animal suffering from a proliferative orinvasion-related disease.

Still further embodiments of the invention include a targeted bindingagent or antibody of the invention for use as a medicament for thetreatment of an animal suffering from tumour cell invasion, cellularmetastasis or tumour growth in an animal.

Still further embodiments of the invention include a targeted bindingagent or antibody of the invention for use as a medicament for thetreatment of an animal suffering from a disease or condition associatedwith B7-H1 expression.

In one embodiment treatment of a proliferative or invasion-relateddisease;

-   -   a neoplastic disease;    -   a non-neoplastic disease;    -   a malignant tumour; or    -   chronic viral infection; or    -   a disease or condition associated with B7-H1 expression,    -   comprises managing, ameliorating, preventing, any of the        aforementioned diseases or conditions.

In one embodiment treatment of a neoplastic disease comprises inhibitionof tumour growth, tumour growth delay, regression of tumour, shrinkageof tumour, increased time to regrowth of tumour on cessation oftreatment, increased time to tumour recurrence, slowing of diseaseprogression.

In one embodiment treatment of a disease or condition associated withB7-H1 expression comprises inhibiting the growth of cells that expressB7-H1.

In some embodiments, following administration of the targeted bindingagent or antibody of the invention, a clearing agent is administered, toremove excess circulating antibody from the blood.

In some embodiments of the invention, the animal to be treated is ahuman.

In some embodiments of the invention, the targeted binding agent is afully human monoclonal antibody.

In some embodiments of the invention, the targeted binding agent isselected from the group consisting of fully human monoclonal antibodies2.7A4, 2.14H9 and 2.9D10.

Embodiments of the invention include a conjugate comprising the targetedbinding agent as described herein, and a therapeutic agent. In someembodiments of the invention, the therapeutic agent is a toxin. In otherembodiments, the therapeutic agent is a radioisotope. In still otherembodiments, the therapeutic agent is a pharmaceutical composition.

In another aspect, a method of selectively killing a cancerous cell in apatient is provided. The method comprises administering a fully humanantibody conjugate to a patient. The fully human antibody conjugatecomprises an antibody that can bind to B7-H1 and an agent. The agent iseither a toxin, a radioisotope, or another substance that will kill acancer cell. The antibody conjugate thereby selectively kills the cancercell.

In one aspect, a conjugated fully human antibody that specifically bindsto B7-H1 is provided. Attached to the antibody is an agent, and thebinding of the antibody to a cell results in the delivery of the agentto the cell. In one embodiment, the above conjugated fully humanantibody binds to an extracellular domain of B7-H1. In anotherembodiment, the antibody and conjugated toxin are internalised by a cellthat expresses B7-H1. In another embodiment, the agent is a cytotoxicagent. In another embodiment, the agent is, for example saporin, orauristatin, Pseudomonas exotoxin, gelonin, ricin, calicheamicin ormaytansine-based immunoconjugates, and the like. In still anotherembodiment, the agent is a radioisotope.

The targeted binding agent or antibody of the invention can beadministered alone, or can be administered in combination withadditional antibodies or chemotherapeutic drugs or radiation therapy ortherapeutic vaccines. For example, a monoclonal, oligoclonal orpolyclonal mixture of B7-H1 antibodies that block B7-H1 mediatedrepression of anti-tumour immunity can be administered in combinationwith a drug shown to inhibit tumour cell proliferation.

According to another aspect of the invention there is provided apharmaceutical composition comprising a targeted binding agent ofantibody of the invention and a pharmaceutically acceptable carrier.

Another embodiment of the invention includes a method of diagnosingdiseases or conditions in which an antibody as disclosed herein isutilised to detect the presence and/or level of B7-H1 in a patient orpatient sample. In one embodiment, the patient sample is blood or bloodserum or urine. In further embodiments, methods for the identificationof risk factors, diagnosis of disease, and staging of disease ispresented which involves the identification of the expression and/oroverexpression of B7-H1 using anti-B7-H1 antibodies. In someembodiments, the methods comprise administering to a patient a fullyhuman antibody conjugate that selectively binds to B7-H1 on a cell. Theantibody conjugate comprises an antibody that specifically binds toB7-H1 and a label. The methods further comprise observing the presenceof the label in the patient. A relatively high amount of the label willindicate a relatively high risk of the disease and a relatively lowamount of the label will indicate a relatively low risk of the disease.In one embodiment, the label is a green fluorescent protein.

The invention further provides methods for assaying for the presenceand/or level of B7-H1 in a patient sample, comprising contacting anantibody as disclosed herein with a biological sample from a patient,and detecting the level of binding between said antibody and B7-H1 insaid sample. In more specific embodiments, the biological sample isblood, plasma or serum.

Another embodiment of the invention includes a method for diagnosing acondition associated with the expression of B7-H1 in a cell bycontacting the serum or a cell with an antibody as disclosed herein, andthereafter detecting the presence of B7-H1. In one embodiment thecondition can be a proliferative or invasion-related disease including,but not limited to, a neoplastic disease.

In another embodiment, the invention includes an assay kit for detectingB7-H1 in mammalian tissues, cells, or body fluids. Such a kit would beuseful to screen for B7-H1-related diseases. The kit includes a targetedbinding agent or antibody of the invention and a means for indicatingthe reaction of the targeted binding agent or antibody with B7-H1, ifpresent. In one embodiment the antibody is a monoclonal antibody. In oneembodiment, the antibody that binds B7-H1 is labeled. In anotherembodiment the antibody is an unlabeled primary antibody and the kitfurther includes a means for detecting the primary antibody. In oneembodiment, the means for detecting includes a labeled second antibodythat is an anti-immunoglobulin. The antibody may be labeled with amarker selected from the group consisting of a fluorochrome, an enzyme,a radionuclide and a radiopaque material.

In some embodiments, the targeted binding agents or antibodies asdisclosed herein can be modified to enhance their capability of fixingcomplement and participating in complement-dependent cytotoxicity (CDC).In other embodiments, the targeted binding agents or antibodies can bemodified to enhance their capability of activating effector cells andparticipating in antibody-dependent cytotoxicity (ADCC). In yet otherembodiments, the targeted binding agents or antibodies can be modifiedboth to enhance their capability of activating effector cells andparticipating in antibody-dependent cytotoxicity (ADCC) and to enhancetheir capability of fixing complement and participating incomplement-dependent cytotoxicity (CDC).

In some embodiments, the targeted binding agents or antibodies asdisclosed herein can be modified to reduce their capability of fixingcomplement and participating in complement-dependent cytotoxicity (CDC).In other embodiments, the targeted binding agents or antibodies can bemodified to reduce their capability of activating effector cells andparticipating in antibody-dependent cytotoxicity (ADCC). In yet otherembodiments, the targeted binding agents or antibodies as disclosedherein can be modified both to reduce their capability of activatingeffector cells and participating in antibody-dependent cytotoxicity(ADCC) and to reduce their capability of fixing complement andparticipating in complement-dependent cytotoxicity (CDC).

In certain embodiments, the half-life of a targeted binding agent orantibody as disclosed herein and of compositions of the invention is atleast about 4 to 7 days. In certain embodiments, the mean half-life of atargeted binding agent or antibody as disclosed herein and ofcompositions of the invention is at least about 2 to 5 days, 3 to 6days, 4 to 7 days, 5 to 8 days, 6 to 9 days, 7 to 10 days, 8 to 11 days,8 to 12, 9 to 13, 10 to 14, 11 to 15, 12 to 16, 13 to 17, 14 to 18, 15to 19, or 16 to 20 days. In other embodiments, the mean half-life of atargeted binding agent or antibody as disclosed herein and ofcompositions of the invention is at least about 17 to 21 days, 18 to 22days, 19 to 23 days, 20 to 24 days, 21 to 25, days, 22 to 26 days, 23 to27 days, 24 to 28 days, 25 to 29 days, or 26 to 30 days. In stillfurther embodiments the half-life of a targeted binding agent orantibody as disclosed herein and of compositions of the invention can beup to about 50 days. In certain embodiments, the half-lives ofantibodies and of compositions of the invention can be prolonged bymethods known in the art. Such prolongation can in turn reduce theamount and/or frequency of dosing of the antibody compositions.Antibodies with improved in vivo half-lives and methods for preparingthem are disclosed in U.S. Pat. No. 6,277,375; and InternationalPublication Nos. WO 98/23289 and WO 97/3461.

In another embodiment, the invention provides an article of manufactureincluding a container. The container includes a composition containing atargeted binding agent or antibody as disclosed herein, and a packageinsert or label indicating that the composition can be used to treatcell adhesion, invasion, angiogenesis, and/or proliferation-relateddiseases, including, but not limited to, diseases characterised by theexpression or overexpression of B7-H1.

In other embodiments, the invention provides a kit for treating diseasesinvolving the expression of B7-H1, comprising a targeted binding agentor antibody as disclosed herein, and instructions to administer themonoclonal antibodies to a subject in need of treatment.

The present invention provides formulation of proteins comprising avariant Fc region. That is, a non naturally occurring Fc region, forexample an Fc region comprising one or more non naturally occurringamino acid residues. Also encompassed by the variant Fc regions ofpresent invention are Fc regions which comprise amino acid deletions,additions and/or modifications.

The serum half-life of proteins comprising Fc regions may be increasedby increasing the binding affinity of the Fc region for FcRn. In oneembodiment, the Fc variant protein has enhanced serum half life relativeto comparable molecule.

In another embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid at one or more positions selected from the group consistingof 239, 330 and 332, as numbered by the EU index as set forth in Kabat.In a specific embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid selected from the group consisting of 239D, 330L and 332E, asnumbered by the EU index as set forth in Kabat. Optionally, the Fcregion may further comprise additional non naturally occurring aminoacid at one or more positions selected from the group consisting of 252,254, and 256, as numbered by the EU index as set forth in Kabat. In aspecific embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid selected from the group consisting of 239D, 330L and 332E, asnumbered by the EU index as set forth in Kabat and at least one nonnaturally occurring amino acid at one or more positions selected fromthe group consisting of 252Y, 254T and 256E, as numbered by the EU indexas set forth in Kabat.

In another embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid at one or more positions selected from the group consistingof 234, 235 and 331, as numbered by the EU index as set forth in Kabat.In a specific embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one non naturally occurringamino acid selected from the group consisting of 234F, 235F, 235Y, 235Eand 331S, as numbered by the EU index as set forth in Kabat. In afurther specific embodiment, an Fc variant of the invention comprisesthe 234F, 235F, and 331S non naturally occurring amino acid residues, asnumbered by the EU index as set forth in Kabat. In another specificembodiment, an Fc variant of the invention comprises the 234F, 235Y, and331S non naturally occurring amino acid residues, as numbered by the EUindex as set forth in Kabat. In another specific embodiment, an Fcvariant of the invention comprises the 234F, 235E, and 331S nonnaturally occurring amino acid residues, as numbered by the EU index asset forth in Kabat. Optionally, the Fc region may further compriseadditional non naturally occurring amino acid at one or more positionsselected from the group consisting of 252, 254, and 256, as numbered bythe EU index as set forth in Kabat. In a specific embodiment, thepresent invention provides an Fc variant, wherein the Fc regioncomprises at least one non naturally occurring amino acid selected fromthe group consisting of 234F, 235F, 235Y, 235E and 331S, as numbered bythe EU index as set forth in Kabat; at least one non naturally occurringamino acid selected from the group consisting of 234F, 235F, and 331S,as numbered by the EU index as set forth in Kabat, and at least one nonnaturally occurring amino acid at one or more positions are selectedfrom the group consisting of 252Y, 254T and 256E, as numbered by the EUindex as set forth in Kabat. As used herein, the “OPT” and “TM”designations are synonymous and are used to describe antibodies of theinvention engineered to introduce the three mutations; L234F and L235Ein the hinge and P331S in the CH2 domain of the IgG molecule toeliminate its ability to trigger antibody-dependent cell-mediatedcytotoxicity and complement-dependent cytotoxicity (Oganesyan V. et al.(2008), Acta Cryst., D64: 700-704).

In another embodiment, the present invention provides an Fc variantprotein formulation, wherein the Fc region comprises at least a nonnaturally occurring amino acid at one or more positions selected fromthe group consisting of 239, 330 and 332, as numbered by the EU index asset forth in Kabat. In a specific embodiment, the present inventionprovides an Fc variant protein formulation, wherein the Fc regioncomprises at least one non naturally occurring amino acid selected fromthe group consisting of 239D, 330L and 332E, as numbered by the EU indexas set forth in Kabat. Optionally, the Fc region may further compriseadditional non naturally occurring amino acid at one or more positionsselected from the group consisting of 252, 254, and 256, as numbered bythe EU index as set forth in Kabat. In a specific embodiment, thepresent invention provides an Fc variant protein formulation, whereinthe Fc region comprises at least one non naturally occurring amino acidselected from the group consisting of 239D, 330L and 332E, as numberedby the EU index as set forth in Kabat and at least one non naturallyoccurring amino acid at one or more positions are selected from thegroup consisting of 252Y, 254T and 256E, as numbered by the EU index asset forth in Kabat.

In another embodiment, the present invention provides an Fc variantprotein formulation, wherein the Fc region comprises at least one nonnaturally occurring amino acid at one or more positions selected fromthe group consisting of 234, 235 and 331, as numbered by the EU index asset forth in Kabat. In a specific embodiment, the present inventionprovides an Fc variant protein formulation, wherein the Fc regioncomprises at least one non naturally occurring amino acid selected fromthe group consisting of 234F, 235F, 235Y, 235E and 331S, as numbered bythe EU index as set forth in Kabat. Optionally, the Fc region mayfurther comprise additional non naturally occurring amino acid at one ormore positions selected from the group consisting of 252, 254, and 256,as numbered by the EU index as set forth in Kabat. In a specificembodiment, the present invention provides an Fc variant proteinformulation, wherein the Fc region comprises at least one non naturallyoccurring amino acid selected from the group consisting of 234F, 235F,235Y, 235E and 331S, as numbered by the EU index as set forth in Kabat;and at least one non naturally occurring amino acid at one or morepositions are selected from the group consisting of 252Y, 254T and 256E,as numbered by the EU index as set forth in Kabat.

Methods for generating non naturally occurring Fc regions are known inthe art. For example, amino acid substitutions and/or deletions can begenerated by mutagenesis methods, including, but not limited to,site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492(1985)), PCR mutagenesis (Higuchi, in “PCR Protocols: A Guide to Methodsand Applications”, Academic Press, San Diego, pp. 177-183 (1990)), andcassette mutagenesis (Wells et al., Gene 34:315-323 (1985)). Preferably,site-directed mutagenesis is performed by the overlap-extension PCRmethod (Higuchi, in “PCR Technology: Principles and Applications for DNAAmplification”, Stockton Press, New York, pp. 61-70 (1989)). Thetechnique of overlap-extension PCR (Higuchi, ibid.) can also be used tointroduce any desired mutation(s) into a target sequence (the startingDNA). For example, the first round of PCR in the overlap-extensionmethod involves amplifying the target sequence with an outside primer(primer 1) and an internal mutagenesis primer (primer 3), and separatelywith a second outside primer (primer 4) and an internal primer (primer2), yielding two PCR segments (segments A and B). The internalmutagenesis primer (primer 3) is designed to contain mismatches to thetarget sequence specifying the desired mutation(s). In the second roundof PCR, the products of the first round of PCR (segments A and B) areamplified by PCR using the two outside primers (primers 1 and 4). Theresulting full-length PCR segment (segment C) is digested withrestriction enzymes and the resulting restriction fragment is clonedinto an appropriate vector. As the first step of mutagenesis, thestarting DNA (e.g., encoding an Fc fusion protein, an antibody or simplyan Fc region), is operably cloned into a mutagenesis vector. The primersare designed to reflect the desired amino acid substitution. Othermethods useful for the generation of variant Fc regions are known in theart (see, e.g., U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425;6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260;6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. PatentPublication Nos. 2004/0002587 and PCT Publications WO 94/29351; WO99/58572; WO 00/42072; WO 02/060919; WO 04/029207: WO 04/099249; WO04/063351).

In some embodiments of the invention, the glycosylation patterns of theantibodies provided herein are modified to enhance ADCC and CDC effectorfunction. See Shields R L et al., (2002) JBC. 277:26733; Shinkawa T etal., (2003) JBC. 278:3466 and Okazaki A et al., (2004) J. Mol. Biol.,336: 1239. In some embodiments, an Fc variant protein comprises one ormore engineered glycoforms, i.e., a carbohydrate composition that iscovalently attached to the molecule comprising an Fc region. Engineeredglycoforms may be useful for a variety of purposes, including but notlimited to enhancing or reducing effector function. Engineeredglycoforms may be generated by any method known to one skilled in theart, for example by using engineered or variant expression strains, byco-expression with one or more enzymes, for example DIN-acetylglucosaminyltransferase III (GnTI11), by expressing a moleculecomprising an Fc region in various organisms or cell lines from variousorganisms, or by modifying carbohydrate(s) after the molecule comprisingFc region has been expressed. Methods for generating engineeredglycoforms are known in the art, and include but are not limited tothose described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davieset al., 20017 Biotechnol Bioeng 74:288-294: Shields et al, 2002, J BiolChem 277:26733-26740: Shinkawa et al., 2003, J Biol Chem 278:3466-3473)U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.10/113,929; PCT WO 00/61739A1: PCT WO 01/292246A1: PCT WO 02/311140A1:PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton,N.J.); GlycoMAb™ glycosylation engineering technology (GLYCARTbiotechnology AG, Zurich, Switzerland). See, e.g., WO 00061739;EA01229125; US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49.

It is also known in the art that the glycosylation of the Fc region canbe modified to increase or decrease effector function (see for examples,Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 2001,Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) U.S.Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929;PCT WO 00/61739A; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO02/30954A; Potillegent™ technology (Biowa, Inc. Princeton, N.J.);GlycoMAb™ glycosylation engineering technology (GLYCART biotechnologyAG, Zurich, Switzerland). Accordingly, in one embodiment the Fc regionsof the antibodies of the invention comprise altered glycosylation ofamino acid residues. In another embodiment, the altered glycosylation ofthe amino acid residues results in lowered effector function. In anotherembodiment, the altered glycosylation of the amino acid residues resultsin increased effector function. In a specific embodiment, the Fc regionhas reduced fucosylation. In another embodiment, the Fc region is afucosylated (see for examples, U.S. Patent Application Publication No.2005/0226867).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the effects of the anti-B7-H1 antibodiesof the invention on T cell proliferation in beads assay.

FIG. 2 is a bar graph showing enhancement of T-cell proliferation byanti-B7-H1 antibodies of the invention in DCMLR assay.

FIG. 3 is a bar graph showing IFN-γ release by anti-B7-H1 antibodies ofthe invention in DCMLR assay.

FIG. 4 is a graph showing 95% confidence interval for anti-B7-H1 IC50 byanti-B7-H1 antibodies of the invention in the human PD1/human B7-H1ligand inhibition assay to evaluate the impact of IgG class switchingand germlining on antibody activity.

FIG. 5 is a line graph showing the results from an ELISA assay that wasperformed to evaluate anti-B7-H1 antibodies of the inventioncross-reactivity to co-modulatory antigens.

FIG. 6 is a bar graph showing the effects of anti-B7-H1 antibodies ofthe invention in the in-vitro Tet-recall assay.

FIG. 7 is a bar graph showing the results form testing agonism activityof anti-B7-H1 antibodies of the invention using the Tet-recall assay.

FIG. 8A/B/C are line graphs showing the effect of anti-B7-H1 antibodiesof the invention on HPAC cells in a mouse xenograft model.

FIG. 9A/B/C are line graphs showing the effect of anti-B7-H1 antibodiesof the invention on A375 cells in a mouse xenograft model.

FIG. 10A/B are line graphs showing the effect of anti-B7-H1 antibodiesof the invention on HPAC cells in a mouse xenograft model.

FIG. 11 is a line graph showing the effect of anti-B7-H1 antibodies ofthe invention on HPAC cells in a mouse xenograft model FIG. 12A/B/C/Dare line graphs showing the effect of anti-B7-H1 antibodies of theinvention on A375 cells in a mouse xenograft model.

FIG. 13A/B are line graphs showing the effect of anti-B7-H1 antibodiesof the invention on A375 cells in a mouse xenograft model with andwithout the presence of T cells.

FIG. 14A/B are line graphs showing the effect of anti-B7-H1 antibodiesof the invention on A375 cells in a mouse xenograft model with andwithout the presence of T cells.

FIG. 15 is a line graph showing the effect of anti-B7-H1 antibodies ofthe invention on A375 cells in a mouse xenograft model.

DEFINITIONS

Unless otherwise defined, scientific and technical terms used hereinshall have the meanings that are commonly understood by those ofordinary skill in the art. Further, unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Generally, nomenclatures utilised in connectionwith, and techniques of, cell and tissue culture, molecular biology, andprotein and oligo- or polynucleotide chemistry and hybridisationdescribed herein are those well known and commonly used in the art.

Standard techniques are used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See for example, e.g., Sambrook et al. Molecular Cloning:A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001)), which is incorporated herein by reference.The nomenclatures utilised in connection with, and the laboratoryprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. Standard techniquesare used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

As utilised in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

An antagonist or inhibitor may be a polypeptide, nucleic acid,carbohydrate, lipid, small molecular weight compound, anoligonucleotide, an oligopeptide, RNA interference (RNAi), antisense, arecombinant protein, an antibody, or fragments thereof or conjugates orfusion proteins thereof. For a review of RNAi see Milhavet O, Gary D S,Mattson M P. (Pharmacol Rev. 2003 December; 55(4):629-48. Review) andantisense (see Opalinska J B, Gewirtz A M. (Sci STKE. 2003 Oct. 28; 2003(206):pe47.)

A compound refers to any small molecular weight compound with amolecular weight of less than about 2000 Daltons.

The term “B7-H1” refers to the human B7-H, B7H1, B7-H1, B7 homolog 1,CD274 antigen, PDCD1L1, PDCD1LG1, PDCD1 ligand 1, PDL1, PD-L1,Programmed cell death 1 ligand 1 precursor, or Programmed death ligand1.

The term “neutralising” or “inhibits” when referring to a targetedbinding agent, such as an antibody, relates to the ability of said agentto eliminate, reduce, or significantly reduce, the activity of a targetantigen. Accordingly, a “neutralising” anti-B7-H1 antibody of theinvention is capable of eliminating or significantly reducing theactivity of B7-H1. A neutralising, antagonising or inhibiting antibodythat specifically binds B7-H1 may, for example, act by blocking thebinding of B7-H1 to its cognate ligands. Ideally, a neutralisingantibody against B7-H1 inhibits B7-H1 mediated repression of T-cellimmunity. A neutralising, antagonising or inhibiting antibody thatspecifically binds B7-H1 may, for example, act by inhibiting binding ofB7-H1 to PD-1 and/or to B7-1.

“Inhibiting the biological activity of B7-H1” encompasses an inhibitionof B7-H1 activity by at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% in comparison with the biological activity in the absence of atargeted binding agent or antibody of the invention.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein, fragments, and analogs are species of the polypeptidegenus. Preferred polypeptides in accordance with the invention comprisethe human heavy chain immunoglobulin molecules and the human kappa lightchain immunoglobulin molecules, as well as antibody molecules formed bycombinations comprising the heavy chain immunoglobulin molecules withlight chain immunoglobulin molecules, such as the kappa or lambda lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof. Preferred polypeptides in accordance with the inventionmay also comprise solely the human heavy chain immunoglobulin moleculesor fragments thereof.

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally occurring.

The term “control sequence” as used herein refers to polynucleotidesequences that are necessary either to effect or to affect theexpression and processing of coding sequences to which they areconnected. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences may includepromoters, enhancers, introns, transcription termination sequences,polyadenylation signal sequences, and 5‘ and’3 untranslated regions. Theterm “control sequences” is intended to include, at a minimum, allcomponents whose presence is essential for expression and processing,and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, orRNA-DNA hetero-duplexes. The term includes single and double strandedforms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non naturally occurring linkages. Oligonucleotides are apolynucleotide subset generally comprising a length of 200 bases orfewer. Preferably, oligonucleotides are 10 to 60 bases in length andmost preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases inlength. Oligonucleotides are usually single stranded, e.g. for probes;although oligonucleotides may be double stranded, e.g. for use in theconstruction of a gene mutant. Oligonucleotides can be either sense orantisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984);Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures ofwhich are hereby incorporated by reference. An oligonucleotide caninclude a label for detection, if desired.

The term “selectively hybridise” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof selectively hybridise to nucleic acid strands underhybridisation and wash conditions that minimise appreciable amounts ofdetectable binding to nonspecific nucleic acids. High stringencyconditions can be used to achieve selective hybridisation conditions asknown in the art and discussed herein. Generally, the nucleic acidsequence homology between the polynucleotides, oligonucleotides, orantibody fragments and a nucleic acid sequence of interest will be atleast 80%, and more typically with preferably increasing homologies ofat least 85%, 90%, 95%, 99%, and 100%.

Stringent hybridization conditions include, but are not limited to,hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate(SSC) (0.9 M NaCl/90 mM NaCitrate, pH 7.0) at about 45° C. followed byone or more washes in 0.2×SSC/0.1% SDS at about 50-65° C., highlystringent conditions such as hybridization to filter-bound DNA in 6×SSCat about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS atabout 60° C., or any other stringent hybridization conditions known tothose skilled in the art (see, for example, Ausubel, F. M. et al., eds.1989 Current Protocols in Molecular Biology, vol. 1, Green PublishingAssociates, Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1 to6.3.6 and 2.10.3).

Two amino acid sequences are “homologous” if there is a partial orcomplete identity between their sequences. For example, 85% homologymeans that 85% of the amino acids are identical when the two sequencesare aligned for maximum matching. Gaps (in either of the two sequencesbeing matched) are allowed in maximising matching; gap lengths of 5 orless are preferred with 2 or less being more preferred. Alternativelyand preferably, two protein sequences (or polypeptide sequences derivedfrom them of at least about 30 amino acids in length) are homologous, asthis term is used herein, if they have an alignment score of at morethan 5 (in standard deviation units) using the program ALIGN with themutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M.O., in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5,National Biomedical Research Foundation (1972)) and Supplement 2 to thisvolume, pp. 1-10. The two sequences or parts thereof are more preferablyhomologous if their amino acids are greater than or equal to 50%identical when optimally aligned using the ALIGN program. It should beappreciated that there can be differing regions of homology within twoorthologous sequences. For example, the functional sites of mouse andhuman orthologues may have a higher degree of homology thannon-functional regions.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence.

In contradistinction, the term “complementary to” is used herein to meanthat the complementary sequence is homologous to all or a portion of areference polynucleotide sequence. For illustration, the nucleotidesequence “TATAC” corresponds to a reference sequence “TATAC” and iscomplementary to a reference sequence “GTATA”.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the comparison window(i.e., the window size), and multiplying the result by 100 to yield thepercentage of sequence identity. The terms “substantial identity” asused herein denotes a characteristic of a polynucleotide or amino acidsequence, wherein the polynucleotide or amino acid comprises a sequencethat has at least 85 percent sequence identity, preferably at least 90to 95 percent sequence identity, more preferably at least 99 percentsequence identity, as compared to a reference sequence over a comparisonwindow of at least 18 nucleotide (6 amino acid) positions, frequentlyover a window of at least 24-48 nucleotide (8-16 amino acid) positions,wherein the percentage of sequence identity is calculated by comparingthe reference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence. As used herein, the twenty conventional amino acids andtheir abbreviations follow conventional usage. See Immunology—ASynthesis (2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., SinauerAssociates, Sunderland, Mass. (1991)), which is incorporated herein byreference. Stereoisomers (e.g., D-amino acids) of the twentyconventional amino acids, unnatural amino acids such asα-,α-disubstituted amino acids. N-alkyl amino acids, lactic acid, andother unconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention. Similarly, unless specified otherwise, theleft-hand end of single-stranded polynucleotide sequences is the 5′ end;the left-hand direction of double-stranded polynucleotide sequences isreferred to as the 5′ direction. The direction of 5′ to 3′ addition ofnascent RNA transcripts is referred to as the transcription direction;sequence regions on the DNA strand having the same sequence as the RNAand which are 5′ to the 5′ end of the RNA transcript are referred to as“upstream sequences”; sequence regions on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the RNA transcriptare referred to as “downstream sequences”. As applied to polypeptides,the term “substantial identity” means that two peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 80 percent sequence identity, preferably atleast 90 percent sequence identity, more preferably at least 95 percentsequence identity, and most preferably at least 99 percent sequenceidentity. Preferably, residue positions that are not identical differ byconservative amino acid substitutions. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Preferred conservative amino acids substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine. Asdiscussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99% sequence identity to theantibodies or immunoglobulin molecules described herein. In particular,conservative amino acid replacements are contemplated. Conservativereplacements are those that take place within a family of amino acidsthat have related side chains. Genetically encoded amino acids aregenerally divided into families: (1) acidic=aspartate, glutamate; (2)basic=lysine, arginine, histidine; (3) non-polar=alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. More preferred families are: serine and threonineare an aliphatic-hydroxy family; asparagine and glutamine are anamide-containing family; alanine, valine, leucine and isoleucine are analiphatic family; and phenylalanine, tryptophan, and tyrosine are anaromatic family. For example, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid willnot have a major effect on the binding function or properties of theresulting molecule, especially if the replacement does not involve anamino acid within a framework site. Whether an amino acid change resultsin a functional peptide can readily be determined by assaying thespecific activity of the polypeptide derivative. Assays are described indetail herein. Fragments or analogs of antibodies or immunoglobulinmolecules can be readily prepared by those of ordinary skill in the art.Preferred amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. Structural and functional domains canbe identified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. Preferably,computerised comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal. Science 253:164 (1991). Thus, the foregoing examples demonstratethat those of skill in the art can recognise sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the antibodies described herein.Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

In general, cysteine residues in proteins are either engaged incysteine-cysteine disulfide bonds or sterically protected from thedisulfide bond formation when they are a part of folded protein region.Disulfide bond formation in proteins is a complex process, which isdetermined by the redox potential of the environment and specializedthiol-disulfide exchanging enzymes (Creighton, Methods Enzymol. 107,305-329, 1984; Houee-Levin, Methods Enzymol. 353, 35-44, 2002). When acysteine residue does not have a pair in protein structure and is notsterically protected by folding, it can form a disulfide bond with afree cysteine from solution in a process known as disulfide shuffling.In another process known as disulfide scrambling, free cysteines mayalso interfere with naturally occurring disulfide bonds (such as thosepresent in antibody structures) and lead to low binding, low biologicalactivity and/or low stability.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterises the parent sequence). Examples of art-recognisedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991), which are each incorporatedherein by reference. Additionally, such methods may be used to makeamino acid substitutions or deletions of one or more variable regioncysteine residues participating in an intrachain disulfide bond togenerate antibody molecules lacking one or more intrachain disulfidebonds.

The term “CDR region” or “CDR” is intended to indicate the hypervariableregions of the heavy and light chains of an antibody which confer theantigen-binding specificity to the antibody. CDRs may be definedaccording to the Kabat system (Kabat, E. A. et al. (1991) Sequences ofProteins of Immunological Interest, 5th Edition. US Department of Healthand Human Services, Public Service, NIH, Washington), and latereditions. An antibody typically contains 3 heavy chain CDRs and 3 lightchain CDRs. The term CDR or CDRs is used here in order to indicate,according to the case, one of these regions or several, or even thewhole, of these regions which contain the majority of the amino acidresidues responsible for the binding by affinity of the antibody for theantigen or the epitope which it recognises.

The third CDR of the heavy chain (HCDR3) has a greater size variability(greater diversity essentially due to the mechanisms of arrangement ofthe genes which give rise to it). It may be as short as 2 amino acidsalthough the longest size known is 26. CDR length may also varyaccording to the length that can be accommodated by the particularunderlying framework. Functionally, HCDR3 plays a role in part in thedetermination of the specificity of the antibody (Segal et al., PNAS,71:4298-4302, 1974, Amit et al., Science, 233:747-753, 1986, Chothia etal., J. Mol. Biol., 196:901-917, 1987, Chothia et al., Nature,342:877-883, 1989, Caton et al., J. Immunol., 144:1965-1968, 1990,Sharon et al., PNAS, 87:4814-4817, 1990, Sharon et al., J. Immunol.,144:4863-4869, 1990, Kabat et al., J. Immunol., 147:1709-1719, 1991).

The term a “set of CDRs” referred to herein comprises CDR1, CDR2 andCDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and HCDR3, and a setof LCDRs refers to LCDR1, LCDR2 and LCDR3.

Variants of the VH and VL domains and CDRs of the present invention,including those for which amino acid sequences are set out herein, andwhich can be employed in targeting binding agents and antibodies forB7-H1 can be obtained by means of methods of sequence alteration ormutation and screening for antigen targeting with desiredcharacteristics. Examples of desired characteristics include but are notlimited to: increased binding affinity for antigen relative to knownantibodies which are specific for the antigen; increased neutralisationof an antigen activity relative to known antibodies which are specificfor the antigen if the activity is known; specified competitive abilitywith a known antibody or ligand to the antigen at a specific molarratio; ability to immunoprecipitate ligand-receptor complex; ability tobind to a specified epitope; linear epitope, e.g. peptide sequenceidentified using peptide-binding scan, e.g. using peptides screened inlinear and/or constrained conformation; conformational epitope, formedby non-continuous residues; ability to modulate a new biologicalactivity of B7-H1, or downstream molecule; ability to bind and/orneutralise B7-H1 and/or for any other desired property. The techniquesrequired to make substitutions within amino acid sequences of CDRs,antibody VH or VL domains and antigen binding sites are available in theart. Variants of antibody molecules disclosed herein may be produced andused in the present invention. Following the lead of computationalchemistry in applying multivariate data analysis techniques to thestructure/property-activity relationships (Wold, et al. Multivariatedata analysis in chemistry. Chemometrics—Mathematics and Statistics inChemistry (Ed.: B. Kowalski), D. Reidel Publishing Company, Dordrecht,Holland, 1984) quantitative activity-property relationships ofantibodies can be derived using well-known mathematical techniques, suchas statistical regression, pattern recognition and classification(Norman et al. Applied Regression Analysis. Wiley-Interscience; 3rdedition (April 1998); Kandel, Abraham & Backer, Eric. Computer-AssistedReasoning in Cluster Analysis. Prentice Hall PTR, (May 11, 1995);Krzanowski, Wojtek. Principles of Multivariate Analysis: A User'sPerspective (Oxford Statistical Science Series, No 22 (Paper)). OxfordUniversity Press; (December 2000); Witten, Ian H. & Frank, Eibe. DataMining: Practical Machine Learning Tools and Techniques with JavaImplementations. Morgan Kaufmann; (Oct. 11, 1999); Denison David G. T.(Editor), Christopher C. Holmes, Bani K. Mallick, Adrian F. M. Smith.Bayesian Methods for Nonlinear Classification and Regression (WileySeries in Probability and Statistics). John Wiley & Sons; (July 2002);Ghose, Arup K. & Viswanadhan, Vellarkad N. Combinatorial Library Designand Evaluation Principles, Software, Tools, and Applications in DrugDiscovery). In some cases the properties of antibodies can be derivedfrom empirical and theoretical models (for example, analysis of likelycontact residues or calculated physicochemical property) of antibodysequence, functional and three-dimensional structures and theseproperties can be considered singly and in combination. An antibodyantigen-binding site composed of a VH domain and a VL domain istypically formed by six loops of polypeptide: three from the light chainvariable domain (VL) and three from the heavy chain variable domain(VH). Analysis of antibodies of known atomic structure has elucidatedrelationships between the sequence and three-dimensional structure ofantibody combining sites. These relationships imply that, except for thethird region (loop) in VH domains, binding site loops have one of asmall number of main-chain conformations: canonical structures. Thecanonical structure formed in a particular loop has been shown to bedetermined by its size and the presence of certain residues at key sitesin both the loop and in framework regions.

This study of sequence-structure relationship can be used for predictionof those residues in an antibody of known sequence, but of an unknownthree-dimensional structure, which are important in maintaining thethree-dimensional structure of its CDR loops and hence maintain bindingspecificity. These predictions can be backed up by comparison of thepredictions to the output from lead optimisation experiments. In astructural approach, a model can be created of the antibody moleculeusing any freely available or commercial package, such as WAM. A proteinvisualisation and analysis software package, such as Insight II(Accelrys, Inc.) or Deep View may then be used to evaluate possiblesubstitutions at each position in the CDR. This information may then beused to make substitutions likely to have a minimal or beneficial effecton activity or confer other desirable properties.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally occurring sequence deduced, for example, froma full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long, morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has at least one ofthe following properties: (1) specific binding to B7-H1, under suitablebinding conditions, (2) ability to block appropriate B7-H1-proteinbinding, or (3) ability to inhibit B7-H1 activity. Typically,polypeptide analogs comprise a conservative amino acid substitution (oraddition or deletion) with respect to the naturally occurring sequence.Analogs typically are at least 20 amino acids long, preferably at least50 amino acids long or longer, and can often be as long as a full-lengthnaturally occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987), which are incorporated herein by reference.Such compounds are often developed with the aid of computerisedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biochemical property or pharmacological activity), such as humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—,by methods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclise the peptide.

As used herein “antibody” and “antibodies” (immunoglobulins) may be anoligoclonal antibody, a polyclonal antibody, a monoclonal antibody(including full-length monoclonal antibodies), a camelised antibody, achimeric antibody, a CDR-grafted antibody, a multi-specific antibody, abi-specific antibody, a catalytic antibody, a chimeric antibody, ahumanized antibody, a fully human antibody, an anti-idiotypic antibodyand antibodies that can be labeled in soluble or bound form as well asfragments, variants or derivatives thereof, either alone or incombination with other amino acid sequences provided by knowntechniques. An antibody may be from any species. An antibody comprises apolypeptide or group of polypeptides that are comprised of at least onebinding domain that is formed from the folding of polypeptide chainshaving three-dimensional binding spaces with internal surface shapes andcharge distributions complementary to the features of an antigenicdeterminant of an antigen. An antibody typically has a tetrameric form,comprising two identical pairs of polypeptide chains, each pair havingone “light” and one “heavy” chain. The variable regions of eachlight/heavy chain pair form an antibody binding site. Native antibodiesare usually heterotetrameric glycoproteins of about 150,000 daltons,composed of two identical light (L) chains and two identical heavy (H)chains. Each light chain is linked to a heavy chain by one covalentdisulfide bond, while the number of disulfide linkages varies betweenthe heavy chains of different immunoglobulin isotypes. Each heavy andlight chain also has regularly spaced intrachain disulfide bridges. Eachheavy chain has at one end a variable domain (VH) followed by a numberof constant domains. Each light chain has a variable domain at one end(VL) and a constant domain at its other end; the constant domain of thelight chain is aligned with the first constant domain of the heavychain, and the light chain variable domain is aligned with the variabledomain of the heavy chain. Light chains are classified as either lambdachains or kappa chains based on the amino acid sequence of the lightchain constant region. The variable domain of a kappa light chain mayalso be denoted herein as VK. The term “variable region” may also beused to describe the variable domain of a heavy chain or light chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains. The variable regions of eachlight/heavy chain pair form an antibody binding site. Such antibodiesmay be derived from any mammal, including, but not limited to, humans,monkeys, pigs, horses, rabbits, dogs, cats, mice, etc.

The term “antibody” or “antibodies” includes binding fragments of theantibodies of the invention, exemplary fragments include single-chainFvs (scFv), single-chain antibodies, single domain antibodies, domainantibodies, Fv fragments, Fab fragments, F(ab′) fragments, F(ab′)2fragments, antibody fragments that exhibit the desired biologicalactivity, disulfide-stabilised variable region (dsFv), dimeric variableregion (Diabody), anti-idiotypic (anti-Id) antibodies (including, e.g.,anti-Id antibodies to antibodies of the invention), intrabodies, linearantibodies, single-chain antibody molecules and multispecific antibodiesformed from antibody fragments and epitope-binding fragments of any ofthe above. In particular, antibodies include immunoglobulin moleculesand immunologically active fragments of immunoglobulin molecules, i.e.,molecules that contain an antigen-binding site. Immunoglobulin moleculescan be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

Digestion of antibodies with the enzyme, papain, results in twoidentical antigen-binding fragments, known also as “Fab” fragments, anda “Fc” fragment, having no antigen-binding activity but having theability to crystallise. Digestion of antibodies with the enzyme, pepsin,results in the a F(ab′)2 fragment in which the two arms of the antibodymolecule remain linked and comprise two-antigen binding sites. TheF(ab′)2 fragment has the ability to crosslink antigen.

“Fv” when used herein refers to the minimum fragment of an antibody thatretains both antigen-recognition and antigen-binding sites. This regionconsists of a dimer of one heavy and one light chain variable domain intight, non-covalent or covalent association. It is in this configurationthat the three CDRs of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer. Collectively,the six CDRs confer antigen-binding specificity to the antibody.However, even a single variable domain (or half of an Fv comprising onlythree CDRs specific for an antigen) has the ability to recognize andbind antigen, although at a lower affinity than the entire binding site.

“Fab” when used herein refers to a fragment of an antibody thatcomprises the constant domain of the light chain and the CH1 domain ofthe heavy chain.

dAb” when used herein refers to a fragment of an antibody that is thesmallest functional binding unit of a human antibodies. A “dAb” is asingle domain antibody and comprises either the variable domain of anantibody heavy chain (VH domain) or the variable domain of an antibodylight chain (VL domain). Each dAb contains three of the six naturallyoccurring CDRs (Ward et al., Binding activities of a repertoire ofsingle immunoglobulin variable domains secreted from Escherichia coli.Nature 341, 544-546 (1989); Holt, et al., Domain antibodies: protein fortherapy, Trends Biotechnol. 21, 484-49 (2003)). With molecular weightsranging from 11 to 15 kDa, they are four times smaller than a fragmentantigen binding (Fab)2 and half the size of a single chain Fv (scFv)molecule.

“Camelid” when used herein refers to antibody molecules are composed ofheavy-chain dimers which are devoid of light chains, but neverthelesshave an extensive antigen-binding repertoire (Hamers-Casterman C,Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa E B, BendahmanN, Hamers R (1993) Naturally occurring antibodies devoid of lightchains. Nature 363:446-448).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (Ward,E. S. et al., (1989) Nature 341, 544-546) the Fab fragment consisting ofVL, VH, CL and CH1 domains; (McCafferty et al (1990) Nature, 348,552-554) the Fd fragment consisting of the VH and CH1 domains; (Holt etal (2003) Trends in Biotechnology 21, 484-490) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989), McCafferty etal (1990) Nature, 348, 552-554, Holt et al (2003) Trends inBiotechnology 21, 484-490], which consists of a VH or a VL domain; (v)isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragmentcomprising two linked Fab fragments (vii) single chain Fv molecules(scFv), wherein a VH domain and a VL domain are linked by a peptidelinker which allows the two domains to associate to form an antigenbinding site (Bird et al, (1988) Science, 242, 423-426, Huston et al,(1988) PNAS USA, 85, 5879-5883); (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804;Holliger, P. (1993) et al, Proc. Natl. Acad. Sci. USA 90 6444-6448). Fv,scFv or diabody molecules may be stabilised by the incorporation ofdisulphide bridges linking the VH and VL domains (Reiter, Y. et al,Nature Biotech, 14, 1239-1245, 1996). Minibodies comprising a scFvjoined to a CH3 domain may also be made (Hu, S. et al, (1996) CancerRes., 56, 3055-3061). Other examples of binding fragments are Fab′,which differs from Fab fragments by the addition of a few residues atthe carboxyl terminus of the heavy chain CH domain, including one ormore cysteines from the antibody hinge region, and Fab′-SH, which is aFab′ fragment in which the cysteine residue(s) of the constant domainsbear a free thiol group.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areresponsible for the binding specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in segments called Complementarity Determining Regions(CDRs) both in the light chain and the heavy chain variable domains. Themore highly conserved portions of the variable domains are called theframework regions (FR). The variable domains of native heavy and lightchains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see, Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are generally not involved directly in antigen binding, but mayinfluence antigen binding affinity and may exhibit various effectorfunctions, such as participation of the antibody in ADCC, CDC, and/orapoptosis.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are associated with its binding toantigen. The hypervariable regions encompass the amino acid residues ofthe “complementarity determining regions” or “CDRs” (e.g., residues24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable domainand residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the heavy chainvariable domain; Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop”(e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Chothia and Lesk, J. Mol. Biol., 196:901-917(1987)). “Framework” or “FR” residues are those variable domain residuesflanking the CDRs. FR residues are present in chimeric, humanized,human, domain antibodies, diabodies, vaccibodies, linear antibodies, andbispecific antibodies.

As used herein, “targeted binding agent”, “targeted binding protein”,“specific binding protein” and like terms refer to an agent, for examplean antibody, or binding fragment thereof, that preferentially binds to atarget site. In one embodiment, the targeted binding agent is specificfor only one target site. In other embodiments, the targeted bindingagent is specific for more than one target site. In one embodiment, thetargeted binding agent may be a monoclonal antibody and the target sitemay be an epitope. A targeted binding agent may comprise at least oneantigen binding domain (e.g. a CDR) of an antibody, wherein said domainis fused or contained within a heterologous protein scaffold, e.g. anon-antibody protein scaffold.

“Binding fragments” of an antibody are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Binding fragments include Fab, Fab′, F(ab′)₂, Fv, dAb and single-chainantibodies. An antibody other than a “bispecific” or “bifunctional”antibody is understood to have each of its binding sites identical. Anantibody substantially inhibits adhesion of a receptor to acounter-receptor when an excess of antibody reduces the quantity ofreceptor bound to counter-receptor by at least about 20%, 40%, 60% or80%, and more usually greater than about 85% (as measured in an in vitrocompetitive binding assay).

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and may, but not always, havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. An antibody is said to specificallybind an antigen when the dissociation constant is ≤1 μM, preferably ≤100nM and most preferably ≤10 nM.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

“Active” or “activity” in regard to a B7-H1 polypeptide refers to aportion of aB7-H1 polypeptide that has a biological or an immunologicalactivity of a native B7-H1 polypeptide. “Biological” when used hereinrefers to a biological function that results from the activity of thenative B7-H1 polypeptide. A preferred B7-H1 biological activityincludes, for example, B7-H1 induced cell proliferation, cell adhesionand invasion.

“Mammal” when used herein refers to any animal that is considered amammal. Preferably, the mammal is human.

“Animal” when used herein encompasses animals considered a mammal.Preferably the animal is human.

The term “patient” includes human and veterinary subjects.

The term “mAb” refers to monoclonal antibody.

“Liposome” when used herein refers to a small vesicle that may be usefulfor delivery of drugs that may include the B7-H1 polypeptide of theinvention or antibodies to such a B7-H1 polypeptide to a mammal.

“Label” or “labeled” as used herein refers to the addition of adetectable moiety to a polypeptide, for example, a radiolabel,fluorescent label, enzymatic label chemiluminescent labeled or abiotinyl group. Radioisotopes or radionuclides may include ³H, ¹⁴C, ¹⁵N,³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, fluorescent labels may includerhodamine, lanthanide phosphors or FITC and enzymatic labels may includehorseradish peroxidase, 0-galactosidase, luciferase, alkalinephosphatase.

Additional labels include, by way of illustration and not limitation:enzymes, such as glucose-6-phosphate dehydrogenase (“G6PDH”),alpha-D-galactosidase, glucose oxydase, glucose amylase, carbonicanhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase andperoxidase; dyes; additional fluorescent labels or fluorescers include,such as fluorescein and its derivatives, fluorochrome, GFP (GFP for“Green Fluorescent Protein”), dansyl, umbelliferone, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine;fluorophores such as lanthanide cryptates and chelates e.g. Europium etc(Perkin Elmer and Cisbio Assays); chemoluminescent labels orchemiluminescers, such as isoluminol, luminol and the dioxetanes;sensitisers; coenzymes; enzyme substrates; particles, such as latex orcarbon particles; metal sol; crystallite; liposomes; cells, etc., whichmay be further labelled with a dye, catalyst or other detectable group;molecules such as biotin, digoxygenin or 5-bromodeoxyuridine; toxinmoieties, such as for example a toxin moiety selected from a group ofPseudomonas exotoxin (PE or a cytotoxic fragment or mutant thereof),Diphtheria toxin or a cytotoxic fragment or mutant thereof, a botulinumtoxin A, B, C, D, E or F, ricin or a cytotoxic fragment thereof e.g.ricin A, abrin or a cytotoxic fragment thereof, saporin or a cytotoxicfragment thereof, pokeweed antiviral toxin or a cytotoxic fragmentthereof and bryodin 1 or a cytotoxic fragment thereof.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient. Otherchemistry terms herein are used according to conventional usage in theart, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)), (incorporatedherein by reference).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%0, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which non-specific cytotoxic cells thatexpress Ig Fc receptors (FcRs) (e.g. Natural Killer (NK) cells,monocytes, neutrophils, and macrophages) recognise bound antibody on atarget cell and subsequently cause lysis of the target cell. The primarycells for mediating ADCC, NK cells, express FcγRIII only, whereasmonocytes express FcγRI, FcγRII and FcγRIII. FcRs expression onhematopoietic cells is summarised in Table 9 on page 464 of Ravetch andKinet, Annu. Rev. Immnol 9:457-92 (1991). To assess ADCC activity of amolecule of interest, an in vitro ADCC assay, such as that described inU.S. Pat. Nos. 5,500,362, or 5,821,337 can be performed. Useful effectorcells for such assays include peripheral blood mononuclear cells (PBMC)and Natural Killer (NK) cells. Alternatively, or additionally, ADCCactivity of the molecule of interest can be assessed in vivo, e.g., inan animal model such as that disclosed in Clynes et al. PNAS (USA)95:652-656 (1988).

“Complement dependent cytotoxicity” and “CDC” refer to the mechanism bywhich antibodies carry out their cell-killing function. It is initiatedby the binding of C1q, a constituent of the first component ofcomplement, to the Fc domain of Igs, IgG or IgM, which are in complexwith antigen (Hughs-Jones, N.C., and B. Gardner. 1979. Mol. Immunol.16:697). C1q is a large, structurally complex glycoprotein of ˜410 kDapresent in human serum at a concentration of 70 μg/ml (Cooper, N. R.1985. Adv. Immunol. 37:151). Together with two serine proteases, C1r andC1s, C1q forms the complex C1, the first component of complement. Atleast two of the N-terminal globular heads of C1q must be bound to theFc of Igs for C1 activation, hence for initiation of the complementcascade (Cooper, N. R. 1985. Adv. Immunol. 37:151).

The term “antibody half-life” as used herein means a pharmacokineticproperty of an antibody that is a measure of the mean survival time ofantibody molecules following their administration. Antibody half-lifecan be expressed as the time required to eliminate 50 percent of a knownquantity of immunoglobulin from the patient's body or a specificcompartment thereof, for example, as measured in serum or plasma, i.e.,circulating half-life, or in other tissues. Half-life may vary from oneimmunoglobulin or class of immunoglobulin to another. In general, anincrease in antibody half-life results in an increase in mean residencetime (MRT) in circulation for the antibody administered.

The term “isotype” refers to the classification of an antibody's heavyor light chain constant region. The constant domains of antibodies arenot involved in binding to antigen, but exhibit various effectorfunctions. Depending on the amino acid sequence of the heavy chainconstant region, a given human antibody or immunoglobulin can beassigned to one of five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM. Several of these classes may be further divided intosubclasses (isotypes), e.g., IgG1 (gamma 1), IgG2 (gamma 2), IgG3 (gamma3), and IgG4 (gamma 4), and IgA1 and IgA2. The heavy chain constantregions that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively. The structures andthree-dimensional configurations of different classes of immunoglobulinsare well-known. Of the various human immunoglobulin classes, only humanIgG1, IgG2, IgG3, IgG4, and IgM are known to activate complement. HumanIgG1, IgG2, IgG3, and IgG4 are known to bind Fc gamma receptors, whichmediate various effector functions including ADCC. Human light chainconstant regions may be classified into two major classes, kappa andlambda.

If desired, the isotype of an antibody that specifically binds B7-H1 canbe switched, for example to take advantage of a biological property of adifferent isotype. For example, in some circumstances it can bedesirable in connection with the generation of antibodies as therapeuticantibodies against B7-H1 that the antibodies be capable of fixingcomplement and participating in complement-dependent cytotoxicity (CDC).There are a number of isotypes of antibodies that are capable of thesame, including, without limitation, the following: murine IgM, murineIgG2a, murine IgG2b, murine IgG3, human IgM, human IgA, human IgG1, andhuman IgG3. In other embodiments it can be desirable in connection withthe generation of antibodies as therapeutic antibodies against B7-H1that the antibodies be capable of binding Fc receptors on effector cellsand participating in antibody-dependent cytotoxicity (ADCC). There are anumber of isotypes of antibodies that are capable of the same,including, without limitation, the following: murine IgG2a, murineIgG2b, murine IgG3, human IgG, and human IgG3. It will be appreciatedthat antibodies that are generated need not initially possess such anisotype but, rather, the antibody as generated can possess any isotypeand the antibody can be isotype switched thereafter using conventionaltechniques that are well known in the art. Such techniques include theuse of direct recombinant techniques (see e.g., U.S. Pat. No.4,816,397), cell-cell fusion techniques (see e.g., U.S. Pat. Nos.5,916,771 and 6,207,418), among others.

By way of example, the anti-B7-H1 antibodies discussed herein are fullyhuman antibodies. If an antibody possessed desired binding to B7-H1, itcould be readily isotype switched to generate a human IgM, human IgG1,or human IgG3 isotype, while still possessing the same variable region(which defines the antibody's specificity and some of its affinity).Such molecule would then be capable of fixing complement andparticipating in CDC and/or be capable of binding to Fc receptors oneffector cells and participating in ADCC.

“Whole blood assays” use unfractionated blood as a source of naturaleffectors. Blood contains complement in the plasma, together withFcR-expressing cellular effectors, such as polymorphonuclear cells(PMNs) and mononuclear cells (MNCs). Thus, whole blood assays allowsimultaneous evaluation of the synergy of both ADCC and CDC effectormechanisms in vitro.

A “therapeutically effective” amount as used herein is an amount thatprovides some improvement or benefit to the subject. Stated in anotherway, a “therapeutically effective” amount is an amount that providessome alleviation, mitigation, and/or decrease in at least one clinicalsymptom. Clinical symptoms associated with the disorders that can betreated by the methods of the invention are well-known to those skilledin the art. Further, those skilled in the art will appreciate that thetherapeutic effects need not be complete or curative, as long as somebenefit is provided to the subject.

Exemplary cancers in humans include a bladder tumour, breast tumour,prostate tumour, basal cell carcinoma, biliary tract cancer, bladdercancer, bone cancer, brain and CNS cancer (e.g., glioma tumour),cervical cancer, choriocarcinoma, colon and rectum cancer, connectivetissue cancer, cancer of the digestive system; endometrial cancer,esophageal cancer; eye cancer; cancer of the head and neck; gastriccancer; intra-epithelial neoplasm; kidney cancer; larynx cancer;leukemia; liver cancer; lung cancer (e.g. small cell and non-smallcell); lymphoma including Hodgkin's and Non-Hodgkin's lymphoma;melanoma; myeloma, neuroblastoma, oral cavity cancer (e.g., lip, tongue,mouth, and pharynx); ovarian cancer; pancreatic cancer, retinoblastoma;rhabdomyosarcoma; rectal cancer, renal cancer, cancer of the respiratorysystem; sarcoma, skin cancer; stomach cancer, testicular cancer, thyroidcancer; uterine cancer, cancer of the urinary system, as well as othercarcinomas and sarcomas.

Exemplary chronic infections in humans include HIV, hepatitis B virus(HBV), and hepatitis C virus (HCV).

The term “and/or” as used herein is to be taken as specific disclosureof each of the two specified features or components with or without theother. For example “A and/or B” is to be taken as specific disclosure ofeach of (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Antibody Structure

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site.

Thus, an intact antibody has two binding sites. Except in bifunctionalor bispecific antibodies, the two binding sites are the same.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called CDRs. The CDRs from the two chains of each pair are alignedby the framework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the definitions of KabatSequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelnyet al. J. Immunol. 148:1547-1553 (1992). Bispecific antibodies do notexist in the form of fragments having a single binding site (e.g., Fab,Fab′, and Fv).

Typically, a VH domain is paired with a VL domain to provide an antibodyantigen-binding site, although a VH or VL domain alone may be used tobind antigen. The VH domain (see Table 9) may be paired with the VLdomain (see Table 13), so that an antibody antigen-binding site isformed comprising both the VH and VL domains.

Human Antibodies and Humanisation of Antibodies

Human antibodies avoid some of the problems associated with antibodiesthat possess murine or rat variable and/or constant regions. Thepresence of such murine or rat derived proteins can lead to the rapidclearance of the antibodies or can lead to the generation of an immuneresponse against the antibody by a patient. In order to avoid theutilisation of murine or rat derived antibodies, fully human antibodiescan be generated through the introduction of functional human antibodyloci into a rodent, other mammal or animal so that the rodent, othermammal or animal produces fully human antibodies.

One method for generating fully human antibodies is through the use ofXenoMouse® strains of mice that have been engineered to contain up tobut less than 1000 kb-sised germline configured fragments of the humanheavy chain locus and kappa light chain locus. See Mendez et al. NatureGenetics 15:146-156 (1997) and Green and Jakobovits J. Med. Med188:483-495 (1998). The XenoMouse® strains are available from Amgen,Inc. (Fremont, Calif., U.S.A).

Such mice, then, are capable of producing human immunoglobulin moleculesand antibodies and are deficient in the production of murineimmunoglobulin molecules and antibodies. Technologies utilised forachieving the same are disclosed in U.S. patent application Ser. No.08/759,620, filed Dec. 3, 1996 and International Patent Application Nos.WO 98/24893, published Jun. 11, 1998 and WO 00/76310, published Dec. 21,2000, the disclosures of which are hereby incorporated by reference. Seealso Mendez et al. Nature Genetics 15:146-156 (1997), the disclosure ofwhich is hereby incorporated by reference.

The production of the XenoMouse® strains of mice is further discussedand delineated in U.S. patent application Ser. No. 07/466,008, filedJan. 12, 1990, Ser. No. 07/610,515, filed Nov. 8, 1990, Ser. No.07/919,297, filed Jul. 24, 1992, Ser. No. 07/922,649, filed Jul. 30,1992, Ser. No. 08/031,801, filed Mar. 15, 1993, Ser. No. 08/112,848,filed Aug. 27, 1993, Ser. No. 08/234,145, filed Apr. 28, 1994, Ser. No.08/376,279, filed Jan. 20, 1995, Ser. No. 08/430,938, filed Apr. 27,1995, Ser. No. 08/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582,filed Jun. 5, 1995, Ser. No. 08/463,191, filed Jun. 5, 1995, Ser. No.08/462,837, filed Jun. 5, 1995, Ser. No. 08/486,853, filed Jun. 5, 1995,Ser. No. 08/486,857, filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun.5, 1995, Ser. No. 08/462,513, filed Jun. 5, 1995, Ser. No. 08/724,752,filed Oct. 2, 1996, Ser. No. 08/759,620, filed Dec. 3, 1996, U.S.Publication 2003/0093820, filed Nov. 30, 2001 and U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2.See alsoEuropean Patent No., EP 0 463 151 B1, grant published Jun. 12, 1996,International Patent Application No., WO 94/02602, published Feb. 3,1994, International Patent Application No., WO 96/34096, published Oct.31, 1996, WO 98/24893, published Jun. 11, 1998, WO 00/76310, publishedDec. 21, 2000. The disclosures of each of the above-cited patents,applications, and references are hereby incorporated by reference intheir entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilised a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more DH genes, one or more J_(H) genes, a mu constant region, andusually a second constant region (preferably a gamma constant region)are formed into a construct for insertion into an animal. This approachis described in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat.Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each toLonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023,010 to Krimpenfortand Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Bernset al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharmInternational U.S. patent application Ser. No. 07/574,748, filed Aug.29, 1990, Ser. No. 07/575,962, filed Aug. 31, 1990, Ser. No. 07/810,279,filed Dec. 17, 1991, Ser. No. 07/853,408, filed Mar. 18, 1992, Ser. No.07/904,068, filed Jun. 23, 1992, Ser. No. 07/990,860, filed Dec. 16,1992, Ser. No. 08/053,131, filed Apr. 26, 1993, Ser. No. 08/096,762,filed Jul. 22, 1993, Ser. No. 08/155,301, filed Nov. 18, 1993, Ser. No.08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699, filed Dec. 10,1993, Ser. No. 08/209,741, filed Mar. 9, 1994, the disclosures of whichare hereby incorporated by reference. See also European Patent No. 0 546073 B1, International Patent Application Nos. WO 92/03918, WO 92/22645,WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175, thedisclosures of which are hereby incorporated by reference in theirentirety. See further Taylor et al., 1992, Chen et al., 1993, Tuaillonet al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al.,(1994), and Tuaillon et al., (1995), Fishwild et al., (1996), thedisclosures of which are hereby incorporated by reference in theirentirety.

Kirin has also demonstrated the generation of human antibodies from micein which, through microcell fusion, large pieces of chromosomes, orentire chromosomes, have been introduced. See European PatentApplication Nos. 773 288 and 843 961, the disclosures of which arehereby incorporated by reference. Additionally, KM™-mice, which are theresult of cross-breeding of Kirin's Tc mice with Medarex's minilocus(Humab) mice have been generated. These mice possess the human IgHtranschromosome of the Kirin mice and the kappa chain transgene of theGenpharm mice (Ishida et al., Cloning Stem Cells, (2002) 4:91-102).

Human antibodies can also be derived by in vitro methods. Suitableexamples include but are not limited to phage display (CAT, Morphosys,Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon),Affimed) ribosome display (CAT), yeast display, and the like.

Preparation of Antibodies

Antibodies, as described herein, were prepared through the utilizationof the XenoMouse® technology, as described below. Such mice are capableof producing human immunoglobulin molecules and antibodies and aredeficient in the production of murine immunoglobulin molecules andantibodies. Technologies utilised for achieving the same are disclosedin the patents, applications, and references disclosed in the backgroundsection herein. In particular, however, a preferred embodiment oftransgenic production of mice and antibodies therefrom is disclosed inU.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996 andInternational Patent Application Nos. WO 98/24893, published Jun. 11,1998 and WO 00/76310, published Dec. 21, 2000, the disclosures of whichare hereby incorporated by reference. See also Mendez et al. NatureGenetics 15:146-156 (1997), the disclosure of which is herebyincorporated by reference.

Through the use of such technology, fully human monoclonal antibodies toa variety of antigens have been produced. Essentially, XenoMouse® linesof mice are immunised with an antigen of interest (e.g. B7-H1),lymphatic cells (such as B-cells) are recovered from the hyper-immunisedmice, and the recovered lymphocytes are fused with a myeloid-type cellline to prepare immortal hybridoma cell lines. These hybridoma celllines are screened and selected to identify hybridoma cell lines thatproduced antibodies specific to the antigen of interest. Provided hereinare methods for the production of multiple hybridoma cell lines thatproduce antibodies specific to B7-H1. Further, provided herein arecharacterisation of the antibodies produced by such cell lines,including nucleotide and amino acid sequence analyses of the heavy andlight chains of such antibodies.

Alternatively, instead of being fused to myeloma cells to generatehybridomas, B cells can be directly assayed. For example, CD19+ B cellscan be isolated from hyperimmune XenoMouse® mice and allowed toproliferate and differentiate into antibody-secreting plasma cells.Antibodies from the cell supernatants are then screened by ELISA forreactivity against the B7-H1 immunogen. The supernatants might also bescreened for immunoreactivity against fragments of B7-H1 to further mapthe different antibodies for binding to domains of functional intereston B7-H1. The antibodies may also be screened other related humanproteins and against the rat, the mouse, and non-human primate, such asCynomolgus monkey, orthologues of B7-H1, the last to determine speciescross-reactivity. B cells from wells containing antibodies of interestmay be immortalised by various methods including fusion to makehybridomas either from individual or from pooled wells, or by infectionwith EBV or transfection by known immortalising genes and then platingin suitable medium. Alternatively, single plasma cells secretingantibodies with the desired specificities are then isolated using aB7-H1-specific hemolytic plaque assay (see for example Babcook et al.,Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)). Cells targeted for lysisare preferably sheep red blood cells (SRBCs) coated with the B7-H1antigen.

In the presence of a B-cell culture containing plasma cells secretingthe immunoglobulin of interest and complement, the formation of a plaqueindicates specific B7-H1-mediated lysis of the sheep red blood cellssurrounding the plasma cell of interest. The single antigen-specificplasma cell in the center of the plaque can be isolated and the geneticinformation that encodes the specificity of the antibody is isolatedfrom the single plasma cell. Using reverse-transcription followed by PCR(RT-PCR), the DNA encoding the heavy and light chain variable regions ofthe antibody can be cloned. Such cloned DNA can then be further insertedinto a suitable expression vector, preferably a vector cassette such asa pcDNA, more preferably such a pcDNA vector containing the constantdomains of immunoglobulin heavy and light chain. The generated vectorcan then be transfected into host cells, e.g., HEK293 cells, CHO cells,and cultured in conventional nutrient media modified as appropriate forinducing transcription, selecting transformants, or amplifying the genesencoding the desired sequences.

As will be appreciated, antibodies as described herein can be expressedin cell lines other than hybridoma cell lines. Sequences encodingparticular antibodies can be used to transform a suitable mammalian hostcell. Transformation can be by any known method for introducingpolynucleotides into a host cell, including, for example packaging thepolynucleotide in a virus (or into a viral vector) and transducing ahost cell with the virus (or vector) or by transfection procedures knownin the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040,4,740,461, and 4,959,455 (which patents are hereby incorporated hereinby reference). The transformation procedure used depends upon the hostto be transformed. Methods for introducing heterologous polynucleotidesinto mammalian cells are well known in the art and includedextran-mediated transfection, calcium phosphate precipitation,polybrene mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalised cell lines available from theNCIMB, including but not limited to Chinese hamster ovary (CHO) cells,HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS),human hepatocellular carcinoma cells (e.g., Hep G2), human epithelialkidney 293 cells (Hek293), and a number of other cell lines. Cell linesof particular preference are selected through determining which celllines have high expression levels and produce antibodies withconstitutive B7-H1 binding properties.

In the cell-cell fusion technique, a myeloma, CHO cell or other cellline is prepared that possesses a heavy chain with any desired isotypeand another myeloma, CHO cell or other cell line is prepared thatpossesses the light chain. Such cells can, thereafter, be fused and acell line expressing an intact antibody can be isolated.

Accordingly, as antibody candidates are generated that meet desired“structural” attributes as discussed above, they can generally beprovided with at least certain of the desired “functional” attributesthrough isotype switching.

In the cell-cell fusion technique, a myeloma, CHO cell or other cellline is prepared that possesses a heavy chain with any desired isotypeand another myeloma, CHO cell or other cell line is prepared thatpossesses the light chain. Such cells can, thereafter, be fused and acell line expressing an intact antibody can be isolated.

Accordingly, as antibody candidates are generated that meet desired“structural” attributes as discussed above, they can generally beprovided with at least certain of the desired “functional” attributesthrough isotype switching.

Antibody Sequences

Embodiments of the invention include the antibodies listed below inTable 1. This table reports the identification number of each antibody,along with the SEQ ID number of the variable domain of the correspondingheavy chain and light chain genes and polypeptides, respectively.

Each antibody sequence has been given an identification number.

TABLE 1 mAb ID SEQ No.: Sequence ID NO: 2.7A4 Nucleotide sequenceencoding the variable 1 region of the heavy chain Amino acid sequenceencoding the variable 2 region of the heavy chain Nucleotide sequenceencoding the variable 6 region of the light chain Amino acid sequenceencoding the variable 7 region of the light chain 2.9D10 Nucleotidesequence encoding the variable 11 region of the heavy chain Amino acidsequence encoding the variable 12 region of the heavy chain Nucleotidesequence encoding the variable 16 region of the light chain Amino acidsequence encoding the variable 17 region of the light chain 2.14H9Nucleotide sequence encoding the variable 21 region of the heavy chainAmino acid sequence encoding the variable 22 region of the heavy chainNucleotide sequence encoding the variable 26 region of the light chainAmino acid sequence encoding the variable 27 region of the light chain2.20A8 Nucleotide sequence encoding the variable 31 region of the heavychain Amino acid sequence encoding the variable 32 region of the heavychain Nucleotide sequence encoding the variable 36 region of the lightchain Amino acid sequence encoding the variable 37 region of the lightchain 3.15G8 Nucleotide sequence encoding the variable 41 region of theheavy chain Amino acid sequence encoding the variable 42 region of theheavy chain Nucleotide sequence encoding the variable 46 region of thelight chain Amino acid sequence encoding the variable 47 region of thelight chain 3.18G1 Nucleotide sequence encoding the variable 51 regionof the heavy chain Amino acid sequence encoding the variable 52 regionof the heavy chain Nucleotide sequence encoding the variable 56 regionof the light chain Amino acid sequence encoding the variable 57 regionof the light chain 2.7A4 Nucleotide sequence encoding the variable 61region of the heavy chain OPT Amino acid sequence encoding the variable62 region of the heavy chain Nucleotide sequence encoding the variable66 region of the light chain Amino acid sequence encoding the variable67 region of the light chain 2.14H9 Nucleotide sequence encoding thevariable 71 OPT region of the heavy chain Amino acid sequence encodingthe variable 72 region of the heavy chain Nucleotide sequence encodingthe variable 76 region of the light chain Amino acid sequence encodingthe variable 77 region of the light chain

Therapeutic Administration and Formulations

Embodiments of the invention include sterile pharmaceutical formulationsof anti-B7-H1 antibodies that are useful as treatments for diseases.Such formulations would inhibit the binding of B7-H1 to one or more ofits cognate ligands, thereby treating pathological conditions where, forexample, serum or tissue B7-H1 is abnormally elevated. Antibodies of theinvention preferably possess adequate affinity to potently inhibit B7-H1activity, or inhibit B7-H1 binding to one or more of its cognateligands, and preferably have an adequate duration of action to allow forinfrequent dosing in humans. A prolonged duration of action will allowfor less frequent and more convenient dosing schedules by alternateparenteral routes such as subcutaneous or intramuscular injection.

Sterile formulations can be created, for example, by filtration throughsterile filtration membranes, prior to or following lyophilisation andreconstitution of the antibody. The antibody ordinarily will be storedin lyophilised form or in solution. Therapeutic antibody compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having an adapter thatallows retrieval of the formulation, such as a stopper pierceable by ahypodermic injection needle.

The route of antibody administration is in accord with known methods,e.g., injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial, intrathecal,inhalation or intralesional routes, direct injection to a tumour site,or by sustained release systems as noted below. The antibody ispreferably administered continuously by infusion or by bolus injection.

An effective amount of antibody to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it ispreferred that the therapist titer the dosage and modify the route ofadministration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer antibody until a dosage isreached that achieves the desired effect. The progress of this therapyis easily monitored by conventional assays or by the assays describedherein.

Antibodies, as described herein, can be prepared in a mixture with apharmaceutically acceptable carrier. This therapeutic composition can beadministered intravenously or through the nose or lung, preferably as aliquid or powder aerosol (lyophilised). The composition can also beadministered parenterally or subcutaneously as desired. Whenadministered systemically, the therapeutic composition should besterile, pyrogen-free and in a parenterally acceptable solution havingdue regard for pH, isotonicity, and stability. These conditions areknown to those skilled in the art. Briefly, dosage formulations of thecompounds described herein are prepared for storage or administration bymixing the compound having the desired degree of purity withpharmaceutically acceptable carriers, excipients, or stabilisers. Suchmaterials are non-toxic to the recipients at the dosages andconcentrations employed, and include buffers such as TRIS HCl,phosphate, citrate, acetate and other organic acid salts; antioxidantssuch as ascorbic acid; low molecular weight (less than about tenresidues) peptides such as polyarginine, proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidinone; amino acids such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium and/or nonionicsurfactants such as TWEEN, PLURONICS or polyethyleneglycol.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice as described in Remington: TheScience and Practice of Pharmacy (20^(th) ed, Lippincott Williams &Wilkens Publishers (2003)). For example, dissolution or suspension ofthe active compound in a vehicle such as water or naturally occurringvegetable oil like sesame, peanut, or cottonseed oil or a syntheticfatty vehicle like ethyl oleate or the like may be desired. Buffers,preservatives, antioxidants and the like can be incorporated accordingto accepted pharmaceutical practice.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thepolypeptide, which matrices are in the form of shaped articles, films ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed Mater. Res., (1981) 15:167-277 andLanger, Chem. Tech., (1982) 12:98-105, or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,(1983) 22:547-556), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLUPRON Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they can denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilisation depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation through disulfideinterchange, stabilisation can be achieved by modifying sulfhydrylresidues, lyophilising from acidic solutions, controlling moisturecontent, using appropriate additives, and developing specific polymermatrix compositions.

Sustained-released compositions also include preparations of crystals ofthe antibody suspended in suitable formulations capable of maintainingcrystals in suspension. These preparations when injected subcutaneouslyor intraperitonealy can produce a sustained release effect. Othercompositions also include liposomally entrapped antibodies. Liposomescontaining such antibodies are prepared by methods known per se: U.S.Pat. No. DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA,(1985) 82:3688-3692; Hwang et al., Proc. Natl. Acad. Sci. USA, (1980)77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949; 142,641;Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324.

The dosage of the antibody formulation for a given patient will bedetermined by the attending physician taking into consideration variousfactors known to modify the action of drugs including severity and typeof disease, body weight, sex, diet, time and route of administration,other medications and other relevant clinical factors. Therapeuticallyeffective dosages can be determined by either in vitro or in vivomethods.

An effective amount of the antibodies, described herein, to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it is preferred for the therapist to titer thedosage and modify the route of administration as required to obtain theoptimal therapeutic effect. A typical daily dosage might range fromabout 0.0001 mg/kg, 0.001 mg/kg, 0.01 mg/kg, 0.1 mg/kg, 1 mg/kg, 10mg/kg to up to 100 mg/kg, 1000 mg/kg, 10000 mg/kg or more, of thepatient's body weight depending on the factors mentioned above. Thedosage may be between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg,0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's bodyweight depending on the factors mentioned above. Typically, theclinician will administer the therapeutic antibody until a dosage isreached that achieves the desired effect. The progress of this therapyis easily monitored by conventional assays or as described herein.

Doses of antibodies of the invention may be repeated and theadministrations may be separated by at least 1 day, 2 days, 3 days, 5days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months,or at least 6 months.

It will be appreciated that administration of therapeutic entities inaccordance with the compositions and methods herein will be administeredwith suitable carriers, excipients, and other agents that areincorporated into formulations to provide improved transfer, delivery,tolerance, and the like. These formulations include, for example,powders, pastes, ointments, jellies, waxes, oils, lipids, lipid(cationic or anionic) containing vesicles (such as Lipofectin™), DNAconjugates, anhydrous absorption pastes, oil-in-water and water-in-oilemulsions, emulsions carbowax (polyethylene glycols of various molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.Any of the foregoing mixtures can be appropriate in treatments andtherapies in accordance with the present invention, provided that theactive ingredient in the formulation is not inactivated by theformulation and the formulation is physiologically compatible andtolerable with the route of administration. See also Baldrick P.“Pharmaceutical excipient development: the need for preclinicalguidance.” Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Wang W.“Lyophilisation and development of solid protein pharmaceuticals.” Int.J. Pharm. 203(1-2):1-60 (2000), Charman W N “Lipids, lipophilic drugs,and oral drug delivery-some emerging concepts.”.J Pharm Sci.89(8):967-78 (2000), Powell et al. “Compendium of excipients forparenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) andthe citations therein for additional information related toformulations, excipients and carriers well known to pharmaceuticalchemists.

Design and Generation of Other Therapeutics

In accordance with the present invention and based on the activity ofthe antibodies that are produced and characterised herein with respectto B7-H1, the design of other therapeutic modalities beyond antibodymoieties is facilitated and disclosed to one skilled in the art. Suchmodalities include, without limitation, advanced antibody therapeutics,such as bispecific antibodies, immunotoxins, radiolabeled therapeutics,and single antibody V domains, antibody-like binding agent based onother than V region scaffolds, single domain antibodies, generation ofpeptide therapeutics, B7-H1 binding domains in novel scaffolds, genetherapies, particularly intrabodies, antisense therapeutics, and smallmolecules.

An antigen binding site may be provided by means of arrangement of CDRson non-antibody protein scaffolds, such as fibronectin or cytochrome Betc. (Haan & Maggos (2004) BioCentury, 12(5): A1-A6; Koide et al. (1998)Journal of Molecular Biology, 284: 1141-1151; Nygren et al. (1997)Current Opinion in Structural Biology, 7: 463-469) or by randomising ormutating amino acid residues of a loop within a protein scaffold toconfer binding specificity for a desired target. Scaffolds forengineering novel binding sites in proteins have been reviewed in detailby Nygren et al. (Nygren et al. (1997) Current Opinion in StructuralBiology, 7: 463-469). Protein scaffolds for antibody mimics aredisclosed in WO/0034784, which is herein incorporated by reference inits entirety, in which the inventors describe proteins (antibody mimics)that include a fibronectin type III domain having at least onerandomised loop. A suitable scaffold into which to graft one or moreCDRs, e.g. a set of HCDRs, may be provided by any domain member of theimmunoglobulin gene superfamily. The scaffold may be a human ornon-human protein. An advantage of a non-antibody protein scaffold isthat it may provide an antigen-binding site in a scaffold molecule thatis smaller and/or easier to manufacture than at least some antibodymolecules. Small size of a binding member may confer usefulphysiological properties, such as an ability to enter cells, penetratedeep into tissues or reach targets within other structures, or to bindwithin protein cavities of the target antigen. Use of antigen bindingsites in non-antibody protein scaffolds is reviewed in Wess, 2004 (Wess,L. In: BioCentury, The Bernstein Report on BioBusiness, 12(42), A1-A7,2004). Typical are proteins having a stable backbone and one or morevariable loops, in which the amino acid sequence of the loop or loops isspecifically or randomly mutated to create an antigen-binding site thatbinds the target antigen. Such proteins include the IgG-binding domainsof protein A from S. aureus, transferrin, albumin, tetranectin,fibronectin (e.g. 10th fibronectin type III domain), lipocalins as wellas gamma-crystalline and other Affilin™ scaffolds (Scil Proteins).Examples of other approaches include synthetic “Microbodies” based oncyclotides—small proteins having intra-molecular disulphide bonds,Microproteins (Versabodies™, Amunix) and ankyrin repeat proteins(DARPins, Molecular Partners).

In addition to antibody sequences and/or an antigen-binding site, atargeted binding agent according to the present invention may compriseother amino acids, e.g. forming a peptide or polypeptide, such as afolded domain, or to impart to the molecule another functionalcharacteristic in addition to ability to bind antigen. Targeted bindingagents of the invention may carry a detectable label, or may beconjugated to a toxin or a targeting moiety or enzyme (e.g. via apeptidyl bond or linker). For example, a targeted binding agent maycomprise a catalytic site (e.g. in an enzyme domain) as well as anantigen binding site, wherein the antigen binding site binds to theantigen and thus targets the catalytic site to the antigen. Thecatalytic site may inhibit biological function of the antigen, e.g. bycleavage.

In connection with the generation of advanced antibody therapeutics,where complement fixation is a desirable attribute, it can be possibleto sidestep the dependence on complement for cell killing through theuse of bispecific antibodies, immunotoxins, or radiolabels, for example.

For example, bispecific antibodies can be generated that comprise (i)two antibodies one with a specificity to B7-H1 and another to a secondmolecule that are conjugated together, (ii) a single antibody that hasone chain specific to B7-H1 and a second chain specific to a secondmolecule, or (iii) a single chain antibody that has specificity to B7-H1and the other molecule. Such bispecific antibodies can be generatedusing techniques that are well known; for example, in connection with(i) and (ii) see e.g., Fanger et al. Immunol Methods 4:72-81 (1994) andWright and Harris, supra. and in connection with (iii) see e.g.,Traunecker et al. Int. J. Cancer (Suppl.) 7:51-52 (1992). In each case,the second specificity can be made as desired. For example, the secondspecificity can be made to the heavy chain activation receptors,including, without limitation, CD16 or CD64 (see e.g., Deo et al.Immunol. Today 18:127 (1997)) or CD89 (see e.g., Valerius et al. Blood90:4485-4492 (1997)).

Antibodies can also be modified to act as immunotoxins utilisingtechniques that are well known in the art. See e.g., Vitetta ImmunolToday 14:252 (1993). See also U.S. Pat. No. 5,194,594. In connectionwith the preparation of radiolabeled antibodies, such modifiedantibodies can also be readily prepared utilising techniques that arewell known in the art. See e.g., Junghans et al. in Cancer Chemotherapyand Biotherapy 655-686 (2d edition, Chafner and Longo, eds., LippincottRaven (1996)). See also U.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827,5,102,990 (RE 35,500), 5,648,471, and 5,697,902. Each immunotoxin orradiolabeled molecule would be likely to kill cells expressing thedesired multimeric enzyme subunit oligomerisation domain.

When an antibody is linked to an agent (e.g., radioisotope,pharmaceutical composition, or a toxin) it is contemplated that theagent possesses a pharmaceutical property selected from the group ofantimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic,alkaloid, COX-2, and antibiotic agents and combinations thereof. Theagent can be selected from the group of nitrogen mustards, ethyleniminederivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acidanalogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs,purine analogs, antimetabolites, antibiotics, enzymes,epipodophyllotoxins, platinum coordination complexes, vinca alkaloids,substituted ureas, methyl hydrazine derivatives, adrenocorticalsuppressants, antagonists, endostatin, taxols, camptothecins,oxaliplatin, doxorubicins and their analogs, and a combination thereof.Examples of toxins further include gelonin, Pseudomonas exotoxin (PE),PE40, PE38, diphtheria toxin, ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, Pseudomonas endotoxin, members of theenediyne family of molecules, such as calicheamicin and esperamicin, aswell as derivatives, combinations and modifications thereof. Chemicaltoxins can also be taken from the group consisting of duocarmycin (see,e.g., U.S. Pat. Nos. 5,703,080 and 4,923,990), methotrexate,doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C,cis-platinum, etoposide, bleomycin and 5-fluorouracil. Examples ofchemotherapeutic agents also include Adriamycin, Doxorubicin,5-Fluorouracil, Cytosine arabinoside (Ara-C), Cyclophosphamide,Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol, Methotrexate,Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide,Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin,Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin,Mitomycins, Esperamicins (see, U.S. Pat. No. 4,675,187), Melphalan, andother related nitrogen mustards. Suitable toxins and chemotherapeuticagents are described in Remington's Pharmaceutical Sciences, 19th Ed.(Mack Publishing Co. 1995), and in Goodman And Gilman's ThePharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co.1985). Other suitable toxins and/or chemotherapeutic agents are known tothose of skill in the art.

Examples of radioisotopes include gamma-emitters, positron-emitters, andx-ray emitters that can be used for localisation and/or therapy, andbeta-emitters and alpha-emitters that can be used for therapy. Theradioisotopes described previously as useful for diagnostics,prognostics and staging are also useful for therapeutics.

Non-limiting examples of anti-cancer or anti-leukemia agents includeanthracyclines such as doxorubicin (adriamycin), daunorubicin(daunomycin), idarubicin, detorubicin, carminomycin, epirubicin,esorubicin, and morpholino and substituted derivatives, combinations andmodifications thereof. Exemplary pharmaceutical agents includecis-platinum, taxol, calicheamicin, vincristine, cytarabine (Ara-C),cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine,chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide,and bleomycin, and derivatives, combinations and modifications thereof.Preferably, the anti-cancer or anti-leukemia is doxorubicin,morpholinodoxorubicin, or morpholinodaunorubicin.

The antibodies of the invention also encompass antibodies that havehalf-lives (e.g., serum half-lives) in a mammal, preferably a human, ofgreater than that of an unmodified antibody. In one embodiment, saidantibody half life is greater than about 15 days, greater than about 20days, greater than about 25 days, greater than about 30 days, greaterthan about 35 days, greater than about 40 days, greater than about 45days, greater than about 2 months, greater than about 3 months, greaterthan about 4 months, or greater than about 5 months. The increasedhalf-lives of the antibodies of the present invention or fragmentsthereof in a mammal, preferably a human, result in a higher serum titerof said antibodies or antibody fragments in the mammal, and thus, reducethe frequency of the administration of said antibodies or antibodyfragments and/or reduces the concentration of said antibodies orantibody fragments to be administered. Antibodies or fragments thereofhaving increased in vivo half-lives can be generated by techniques knownto those of skill in the art. For example, antibodies or fragmentsthereof with increased in vivo half-lives can be generated by modifying(e.g., substituting, deleting or adding) amino acid residues identifiedas involved in the interaction between the Fc domain and the FcRnreceptor (see, e.g., International Publication Nos. WO 97/34631 and WO02/060919, which are incorporated herein by reference in theirentireties). Antibodies or fragments thereof with increased in vivohalf-lives can be generated by attaching to said antibodies or antibodyfragments polymer molecules such as high molecular weightpolyethyleneglycol (PEG). PEG can be attached to said antibodies orantibody fragments with or without a multifunctional linker eitherthrough site-specific conjugation of the PEG to the N- or C-terminus ofsaid antibodies or antibody fragments or via epsilon-amino groupspresent on lysine residues. Linear or branched polymer derivatisationthat results in minimal loss of biological activity will be used. Thedegree of conjugation will be closely monitored by SDS-PAGE and massspectrometry to ensure proper conjugation of PEG molecules to theantibodies. Unreacted PEG can be separated from antibody-PEG conjugatesby, e.g., size exclusion or ion-exchange chromatography.

As will be appreciated by one of skill in the art, in the aboveembodiments, while affinity values can be important, other factors canbe as important or more so, depending upon the particular function ofthe antibody. For example, for an immunotoxin (toxin associated with anantibody), the act of binding of the antibody to the target can beuseful; however, in some embodiments, it is the internalisation of thetoxin into the cell that is the desired end result. As such, antibodieswith a high percent internalisation can be desirable in thesesituations. Thus, in one embodiment, antibodies with a high efficiencyin internalisation are contemplated. A high efficiency ofinternalisation can be measured as a percent internalised antibody, andcan be from a low value to 100%. For example, in varying embodiments,0.1-5, 5-10, 10-20, 20-30, 30-40, 40-45, 45-50, 50-60, 60-70, 70-80,80-90, 90-99, and 99-100% can be a high efficiency. As will beappreciated by one of skill in the art, the desirable efficiency can bedifferent in different embodiments, depending upon, for example, theassociated agent, the amount of antibody that can be administered to anarea, the side effects of the antibody-agent complex, the type (e.g.,cancer type) and severity of the problem to be treated.

In other embodiments, the antibodies disclosed herein provide an assaykit for the detection of B7-H1 expression in mammalian tissues or cellsin order to screen for a disease or disorder associated with changes inexpression of B7-H1. The kit comprises an antibody that binds B7-H1 andmeans for indicating the reaction of the antibody with the antigen, ifpresent.

In some embodiments, an article of manufacture is provided comprising acontainer, comprising a composition containing an antibody thatspecifically binds B7-H1, and a package insert or label indicating thatthe composition can be used to treat disease mediated by B7-H1expression. Preferably a mammal and, more preferably, a human, receivesthe antibody that specifically binds B7-H1.

Combinations

The anti-tumour treatment defined herein may be applied as a soletherapy or may involve, in addition to the compounds of the invention,conventional surgery, bone marrow and peripheral stem celltransplantations or radiotherapy or chemotherapy. Such chemotherapy mayinclude one or more of the following categories of anti tumour agents:

(i) other antiproliferative/antineoplastic drugs and combinationsthereof, as used in medical oncology, such as alkylating agents (forexample cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogenmustard, melphalan, chlorambucil, busulphan, temozolamide andnitrosoureas); antimetabolites (for example gemcitabine and antifolatessuch as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed,methotrexate, cytosine arabinoside, and hydroxyurea); anti-tumorantibiotics (for example anthracyclines like adriamycin, bleomycin,doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin); antimitotic agents (for example vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like taxol and taxotere and polokinase inhibitors); andtopoisomerase inhibitors (for example epipodophyllotoxins like etoposideand teniposide, amsacrine, topotecan and camptothecin);(ii) cytostatic agents such as antioestrogens (for example tamoxifen,fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene),antiandrogens (for example bicalutamide, flutamide, nilutamide andcyproterone acetate), LHRH antagonists or LHRH agonists (for examplegoserelin, leuprorelin and buserelin), progestogens (for examplemegestrol acetate), aromatase inhibitors (for example as anastrozole,letrozole, vorazole and exemestane) and inhibitors of 5α-reductase suchas finasteride;(iii) anti-invasion agents (for example c-Src kinase family inhibitorslike4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-tetrahydropyran-4-yloxyquinazoline(AZD0530; International Patent Application WO 01/94341) andN-(2-chloro-6-methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-ylamino}thiazole-5-carboxamide(dasatinib, BMS-354825; J. Med. Chem., 2004, 47, 6658-6661), andmetalloproteinase inhibitors like marimastat, inhibitors of urokinaseplasminogen activator receptor function or inhibitors of cathepsinactivity, inhibitors of serine proteases for example matriptase, hepsin,urokinase and inhibitors of integrin αvβ6 function.(iv) cytotoxic agents such as fludarabine, 2-chlorodeoxyadenosine,chlorambucil or doxorubicin and combination thereof such asFludarabine+cyclophosphamide, CVP:cyclophosphamide+vincristine+prednisone, ACVBP:doxorubicin+cyclophosphamide+vindesine+bleomycin+prednisone, CHOP:cyclophosphamide+doxorubicin+vincristine+prednisone, CNOP:cyclophosphamide+mitoxantrone+vincristine+prednisone, m-BACOD:methotrexate+bleomycin+doxorubicin+cyclophosphamide+vincristine+dexamethasone+leucovorin,MACOP-B:methotrexate+doxorubicin+cyclophosphamide+vincristine+prednisone fixeddose+bleomycin+leucovorin, or ProMACE CytaBOM:prednisone+doxorubicin+cyclophosphamide+etoposide+cytarabine+bleomycin+vincristine+methotrexate+leucovorin.(v) inhibitors of growth factor function: for example such inhibitorsinclude growth factor antibodies and growth factor receptor antibodies(for example the anti-erbB2 antibody trastuzumab [Herceptin™], theanti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab[Erbitux, C225] and any growth factor or growth factor receptorantibodies disclosed by Stern et al. Critical reviews inoncology/haematology, 2005, Vol. 54, pp 11-29); such inhibitors alsoinclude tyrosine kinase inhibitors, for example inhibitors of theepidermal growth factor family (for example EGFR family tyrosine kinaseinhibitors such asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib, ZD1839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI-774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine(CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib,inhibitors of the hepatocyte growth factor family, inhibitors of theplatelet-derived growth factor family such as imatinib, inhibitors ofserine/threonine kinases (for example Ras/Raf signalling inhibitors suchas farnesyl transferase inhibitors, for example sorafenib (BAY43-9006)), inhibitors of cell signalling through MEK and/or AKT kinases,inhibitors of the hepatocyte growth factor family, c-kit inhibitors, ablkinase inhibitors, IGF receptor (insulin-like growth factor) kinaseinhibitors; aurora kinase inhibitors (for example AZD1152, PH739358,VX-680, MLN8054, R763, MP235, MP529, VX-528 AND AX39459) and cyclindependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors, andinhibitors of survival signaling proteins such as Bcl-2, Bcl-XL forexample ABT-737;(vi) antiangiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, [for example the anti-vascularendothelial cell growth factor antibody bevacizumab (Avastin™)] and VEGFreceptor tyrosine kinase inhibitors such as4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474; Example 2 within WO 01/32651),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO98/35985) and SU11248 (sunitinib; WO 01/60814), compounds such as thosedisclosed in International Patent Applications WO97/22596, WO 97/30035,WO 97/32856, WO 98/13354, WO00/47212 and WO01/32651, anti-vascularendothelial growth factor receptor antibodies such anti-KDR antibodiesand anti-fit1 antibodies) and compounds that work by other mechanisms(for example linomide, inhibitors of integrin αvβ3 function andangiostatin)] or colony stimulating factor 1 (CSF1) or CSF1 receptor;Additional details on AZD2171 may be found in Wedge et al (2005) CancerResearch. 65(10):4389-400. Additional details on AZD6474 may be found inRyan & Wedge (2005) British Journal of Cancer. 92 Suppl 1:S6-13. Bothpublications are herein incorporated by reference in their entireties.(vii) vascular damaging agents such as Combretastatin A4 and compoundsdisclosed in International Patent Applications WO 99/02166, WO 00/40529,WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;(viii) antisense therapies, for example those which are directed to thetargets listed above, such as ISIS 2503, an anti-ras antisense or G3139(Genasense), an anti bcl2 antisense;(ix) gene therapy approaches, including for example approaches toreplace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2,GDEPT (gene-directed enzyme pro-drug therapy) approaches such as thoseusing cytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy such as multi-drug resistance gene therapy;(x) immunotherapy approaches, including for example treatment withAlemtuzumab (campath-1H™), a monoclonal antibody directed at CD52, ortreatment with antibodies directed at CD22, ex vivo and in vivoapproaches to increase the immunogenicity of patient tumour cells,transfection with cytokines such as interleukin 2, interleukin 4 orgranulocyte macrophage colony stimulating factor, approaches to decreaseT cell anergy such as treatment with monoclonal antibodies inhibitingCTLA-4 function, approaches using transfected immune cells such ascytokine transfected dendritic cells, approaches using cytokinetransfected tumour cell lines and approaches using anti idiotypicantibodies, adoptive T-cell transfer using T-cells that have beennon-specifically activated or targeted to a specific antigen of interestex vivo;(xi) Vaccination approaches, including for example treatment with avaccine directed against a specific viral infection such as HIV or HBV,or treatment with a vaccine directed against a specific tumour antigen;(xii) inhibitors of protein degradation such as proteasome inhibitorsuch as Velcade (bortezomid); and(xiii) biotherapeutic therapeutic approaches for example those which usepeptides or proteins (such as antibodies or soluble external receptordomain constructions) which either sequester receptor ligands, blockligand binding to receptor or decrease receptor signalling (e.g. due toenhanced receptor degradation or lowered expression levels).

In one embodiment the anti-tumour treatment defined herein may involve,in addition to the compounds of the invention, treatment with otherantiproliferative/antineoplastic drugs and combinations thereof, as usedin medical oncology, such as alkylating agents (for example cis-platin,oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan,chlorambucil, busulphan, temozolamide and nitrosoureas); antimetabolites(for example gemcitabine and antifolates such as fluoropyrimidines like5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosinearabinoside, and hydroxyurea); anti-tumor antibiotics (for exampleanthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin,epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin);antimitotic agents (for example vinca alkaloids like vincristine,vinblastine, vindesine and vinorelbine and taxoids like taxol andtaxotere and polokinase inhibitors); and topoisomerase inhibitors (forexample epipodophyllotoxins like etoposide and teniposide, amsacrine,topotecan and camptothecin).

Such conjoint treatment may be achieved by way of the simultaneous,sequential or separate dosing of the individual components of thetreatment. Such combination products employ the compounds of thisinvention, or pharmaceutically acceptable salts thereof, within thedosage range described hereinbefore and the other pharmaceuticallyactive agent within its approved dosage range.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the teachings herein.

Example 1: Expression of Recombinant Human B7-H1

The human B7-H1 cDNA (Dong, H. et al., 1999, Nat. Med. 5:1365-1369) wasamplified from Image clone 7262208 (ATCC) using polymerase chainreaction (PCR) and then cloned into the Nhe 1 and EcoR1 sites ofpcr3.1Bid vector. This construct was lipofected into CHO cells (AmericanType Tissue Collection, catalog #CCL-61) and the expression on the cellsurface was confirmed by fluorescent activated cell sorting (FACS)analysis.

The extracellular domain of human B7-H1 (amino acid residues 1-239)fused to the Fc region of human IgG1 was purchased from R&D SystemsInc., catalog #156-B7-100.

Example 2: Immunisation and Titering

Immunisation

Monoclonal antibodies against human B7-H1 were developed by sequentiallyimmunizing XenoMouse® mice (XenoMouse strains: XMG2 (IgG2 kappa/lambda)and XMG4 (IgG4 kappa/lambda) Amgen, Inc. Vancouver, British Columbia,Canada) with either 5-10 ug of the B7-H1/Fc chimera protein or 1-2×10(6)CHO cells expressing recombinant human B7-H1 as described in Example 1.

Immunisations were conducted according to the methods disclosed in U.S.patent application Ser. No. 08/759,620, filed Dec. 3, 1996 andInternational Patent Application Nos. WO 98/24893, published Jun. 11,1998 and WO 00/76310, published Dec. 21, 2000, the disclosures of whichare hereby incorporated by reference. The immunisation programs aresummarised in Table 2.

Selection of Animals for Harvest by Titer

Titres of antibodies in sera from the immunised mice were determined inan ELISA assay. Plates (Corning Costar, cat #3368) were coated with thehuman B7-H1/Fc protein (R&D Systems Inc., catalog #156-B7-100).B7-H1-specific antibodies were detected with mouse anti-human IgGantibody and goat anti-mouse IgG Fc antibody conjugated to horseradishperoxidase. Typically, five animals with the highest titres within eachimmunization cohort were selected for lymphocyte isolation andgeneration of hybridomas.

TABLE 2 Summary of Immunisation Programs No of Immunisation GroupImmunogen Strain mice routes 1 Recombinant human B7- IgG2 10 IP/SC,twice/wk, for H1/Fc protein 5 weeks 2 Recombinant human B7- IgG4 10IP/SC, twice/wk, for H1/Fc protein 5 weeks 3 CHO cells expressing IgG210 IP/SC, twice/wk, for human B7-H1 5 weeks 4 CHO cells expressing IgG410 IP/SC, twice/wk, for human B7-H1 5 weeks “IP” refers to“intraperitoneal” and “SC” refers to “subcutaneous”

Example 3: Recovery of Lymphocytes, B-Cell Isolations, Fusions andGeneration of Hybridomas

Immunised mice were sacrificed by cervical dislocation, and the draininglymph nodes harvested and pooled from each cohort. The lymphoid cellswere dissociated by grinding in DMEM to release the cells from thetissues and the cells were suspended in DMEM. The cells were counted,and 0.9 ml DMEM per 100 million lymphocytes added to the cell pellet toresuspend the cells gently but completely. Using 100 μl of CD90+magnetic beads per 100 million cells, the cells were labeled byincubating the cells with the magnetic beads at 4° C. for 15 minutes.The magnetically labeled cell suspension containing up to 10⁸ positivecells (or up to 2×10⁹ total cells) was loaded onto a LS+ column and thecolumn washed with DMEM. The total effluent was collected as theCD90-negative fraction (most of these cells were expected to be Bcells).

The fusion was performed by mixing washed enriched B cells from aboveand nonsecretory myeloma P3X63Ag8.653 cells purchased from ATCC, cat.#CRL 1580 (Kearney et al, J. Immunol. 123, 1979, 1548-1550) at a ratioof 1:1. The cell mixture was gently pelleted by centrifugation at 800×g.After complete removal of the supernatant, the cells were treated with2-4 mL of Pronase solution (CalBiochem, cat. #53702; 0.5 mg/ml in PBS)for no more than 2 minutes. Then 3-5 ml of FBS was added to stop theenzyme activity and the suspension was adjusted to 40 ml total volumeusing electro cell fusion solution, (ECFS, 0.3M Sucrose, Sigma, Cat#S7903, 0.1 mM Magnesium Acetate, Sigma, Cat #M2545, 0.1 mM CalciumAcetate, Sigma, Cat #C4705). The supernatant was removed aftercentrifugation and the cells were resuspended in 40 ml ECFS. This washstep was repeated and the cells again were resuspended in ECFS to aconcentration of 2×10⁶ cells/ml.

Electro-cell fusion was performed using a fusion generator (modelECM2001, Genetronic, Inc., San Diego, Calif.). The fusion chamber sizeused was 2.0 ml, using the following instrument settings:

-   -   Alignment condition: voltage: 50 V, time: 50 sec.    -   Membrane breaking at: voltage: 3000 V, time: 30 ρsec    -   Post-fusion holding time: 3 sec

After ECF, the cell suspensions were carefully removed from the fusionchamber under sterile conditions and transferred into a sterile tubecontaining the same volume of Hybridoma Culture Medium (DMEM, JRHBiosciences), 15% FBS (Hyclone), supplemented with L-glutamine,pen/strep, OPI (oxaloacetate, pyruvate, bovine insulin) (all from Sigma)and IL-6 (Boehringer Mannheim). The cells were incubated for 15-30minutes at 37° C., and then centrifuged at 400×g (1000 rpm) for fiveminutes. The cells were gently resuspended in a small volume ofHybridoma Selection Medium (Hybridoma Culture Medium supplemented with0.5× HA (Sigma, cat. #A9666)), and the volume adjusted appropriatelywith more Hybridoma Selection Medium, based on a final plating of 5×10⁶B cells total per 96-well plate and 200 μl per well. The cells weremixed gently and pipetted into 96-well plates and allowed to grow. Onday 7 or 10, one-half the medium was removed, and the cells re-fed withHybridoma Selection Medium.

Hybridomas were grown as routine in the selective medium. Exhaustivesupernatants collected from the hybridomas were tested in various assaysas described in Examples 4-5. Just to note, antibodies that begin with a3 digit, e.g., 3.15G8, are IgG4 and antibodies that begin with a 2,e.g., 2.9D10, are IgG2.

Example 4: Binding to Cell Bound Human and Cynomolgus Monkey B7-H1

Supernatants collected from hybridoma cells were tested to assess theability of the secreted antibodies to bind to 293T cells transientlyexpressing either full length human or cynomolgus monkey B7-H1. Amock-transfected 293T cell line was used as a negative control. Cellsdiluted in PBS containing 2% FBS were seeded at a density of 2500-3000expressing and 15000-17500 mock transfected cells in 40 μl/well in 384well plates (Corning Costar, catalog #3712). Immediately after plating,10 μl/well of hybridoma supernatants were added and plates incubated for1.5 hr at room temperature. Next, 10 μl/well of Cy5-conjugated goatanti-human IgG Fc (700 ng/ml, Jackson Immunoresearch, catalog#109-175-098) were added and plates incubated for 3 h at roomtemperature prior to reading the fluorescence signal on the FMAT 8200instrument (Applied Biosystems). The results for 6 hybridomasupernatants are shown in Table 3.

TABLE 3 Binding of hybridoma supernatants to cell-bound human orcynomologus B7-H1 Human B7-H1 binding Cynomolgus monkey B7-H1 bindingAntibody ID Count FL1 FL1xcount Count FL1 FL1xcount 2.9D10 22 7176157872 54 5526.9 298454 2.14H9 31 10289 318949 56 6777.9 379561 3.15G822 8811 193850 64 8647.1 553414 2.20A8 34 10283 349622 65 7930.5 5154842.7A4 37 6975.9 258108 59 8697.7 513164 3.18G1 21 10862 228106 6610336.3 682195

Example 5: Inhibition of B7-H1/PD-1 Receptor-Ligand Binding

To determine the relative potency of the antibody containingsupernatants, their ability to inhibit the binding of human PD-1/Fcprotein to human B7-H1 expressed on surface of CHO cells was evaluated.25000 cells/well were plated in 50 μl of media into wells of a 384-welltissue culture plate (Corning Costar, catalog #3712). Next day, 50μl/well of diluted (1:5) hybridoma supernatant was added and plates wereincubated at 4 C for 1 hour on a shaker. Biotinylated human PD-1/Fcprotein (R&D Systems, catalog #1086-PD) was added to a finalconcentration of 1.25 ug/ml and plates were incubated at 4 C for 1 houron a shaker. Cells were washed and fixed in 100 ul of PBS containing3.7% formaldehyde and 3% bovine serum albumin for 20 min at roomtemperature. Cells were washed and incubated in 100 ul of PBS containing0.6% H2O2 and 3% bovine serum albumin for 10 min at room temperature.Cells were washed and incubated in 50 μl of horseradishperoxidase-conjugated streptavidin diluted at 1:4000 for 30 min at 4° C.Cells were washed before signal detection. Data are represented as thepercentage of (ODmax-ODmin), where ODmax is the average value obtainedwith the cells incubated with irrelevant hybridoma supernatants in thepresence of biotin-conjugated human PD-1/Fc protein and ODmin is theaverage value obtained with the cells incubated with irrelevanthybridoma supernatants in the absence of biotin-conjugated human PD-1/Fcprotein. 0% of maximum response indicates 100% inhibition of B7-H1/PD-1binding by a hybridoma supernatant (Table 4).

TABLE 4 Inhibition of human PD1 binding to human B7-H1 expressing cellsby hybridoma supernatants PD1-Fc binding to CHO- Antibody ID huB7-H1cells (% of max) 2.9D10 −5 2.14H9 5 3.15G8 4 2.20A8 0 2.7A4 0 3.18G1 5

Example 6: Binding of Purified Anti-B7-H1 Antibodies to Human B7-H1,Human B7-DC, Mouse B7-H1

The ability of the purified antibodies to bind to human B7-H1, B7-DC,mouse B7H1 and cynomolgus monkey B7-H1 was determined by FACS analysis.Briefly, 293T cells were either mock-transfected or transientlytransfected with either human B7-H1 or human B7-DC using Lipofectamine2000 (Invitrogen, catalog #11668). Mouse J558 cells expressing mouseB7-H1 were obtained from ATCC (catalog #TIB-6). Cells were resuspendedin PBS containing 2% FBS (FACS buffer) and seeded at 50000 cells/wellinto V-bottomed plates. Anti-B7-H1 and isotype control antibodiesdiluted in FACS buffer were added at a final concentration of 5 μg/mland plates were incubated for 1 h at 4° C. After washing with FACSbuffer, goat anti-human Fc Cy5 (5 μg/ml, Jackson Immunoresearch, catalog#109-175-098) and 7-AAD (5 μg/ml) were added and plates were incubatedfor 15 min at 4° C. before being washed again with FACS buffer and beingread on a FACSCalibur instrument. Table 5 shows ability of purifiedantibodies (5 μg/ml) to bind to 293T cells transfected with human B7-H.None of the selected antibodies bound to 293T cells transfected withhuman B7-DC or to J558 cells that express mouse B7-H1. Mouse anti-humanB7-DC (PD-L2) antibody (R&D systems cat #MAB1224, detected with goatanti-mouse Fc Cy5, Jackson Immunoresearch) was used as a positivecontrol for B7-DC expression. PE-conjugated rat anti-mouse B7-H1antibody (eBioscience, clone MH5, detected with goat anti-rat Fc Cy5,Jackson Immunoresearch) was used as a positive control for mouse B7-H1expression.

TABLE 5 Binding of purified anti-B7-H1 antibodies to cell-bound humanB7-H1, mouse B7-H1 or human B7-DC Human(h)B7- Human(h)B7- H1/293TDC/293T 293T J558 Ab ID (geo mean) (geo mean) (geo mean) (geo mean2.9D10 463.3 8.7 8.9 3.2 2.14H9 792.1 11.4 9.9 3.0 3.15G8 1183.3 10.19.4 3.2 2.20A8 462.8 9.5 8.4 3.3 2.7A4 753.8 10.5 9.4 3.2 3.18G1 1539.69.7 10.5 3.2 IgG2 7.6 9.5 9.1 3.1 IgG4 10.7 8.8 9.3 3.2

Example 7: Binding of Purified Human Anti-B7-H1 Antibodies to StimulatedHuman and Cynomolgus Monkey T Cells

96-well high binding plates were incubated with 100 ul/well of anti-CD3antibody diluted at 1 ug/ml in PBS (OKT3 clone, eBioscience, catalog#160037) at 4 C overnight. Human T cells were isolated from frozenleukopack using T cell enrichment kit (StemCell Technologies, catalog#19051). Anti-CD3 mAb coated plates were washed with PBS and purified Tcells were added in 200 ul of ICM media at 360000 cells/well andcultured for 72 hours. T cells were then harvested, washed in FACSbuffer and mixed with diluted purified anti-B7-H1 antibodies orirrelevant human IgG2 or IgG4 antibodies at final concentration of 1ug/ml in 96-well V-bottom assay plate (50 ul/well). After 2 hoursincubation at 4 C, T cells were washed twice in FACS buffer and thenstained with Cy5-conjugated goat anti-human IgG Fc antibody (5 ug/ml,Jackson Immunoresearch, catalog #109-175-098) and 7-AAD (10 ug/ml).Cells were incubated for 30 min at 4° C. before being washed again withFACS buffer and being read on a FACSCalibur instrument. Live lymphocytepopulation was selected for analysis based on forward and side scatteras well as negative staining for 7-AAD.

For activation of cynomolgus monkey T cells 96-well high binding plateswere incubated with 100 ul/well of goat anti-mouse IgG Fc antibodydiluted at 1 ug/ml in PBS at 4 C overnight. Plates were washed with PBSand incubated with anti-CD3 antibody diluted at 1 ug/ml in ICM media(clone SP-34, BD catalog #556610) at 37 C for 2 hours. Cynomolgus monkeyPBMC were isolated from peripheral blood (Bioreclamation, catalog#CYNWBCPT). Anti-CD3 mAb coated plates were washed with PBS and isolatedPBMC were added in 200 ul of ICM media at ˜200000 cells/well andcultured for 72 hours. Cells were then harvested, washed in FACS bufferand mixed with diluted purified anti-B7-H1 antibodies or irrelevanthuman IgG2 or IgG4 antibodies at a final concentration of 1 ug/ml in96-well V-bottom assay plate (50 ul/well). After 2 hours incubation at 4C, cells were washed twice in FACS buffer and then stained withCy5-conjugated goat anti-human IgG Fc antibody (5 ug/ml, JacksonImmunoresearch, catalog #109-175-098), FITC-conjugated anti-CD3 antibody(diluted 1:25, Biospecialty) and 7-AAD (10 ug/ml). Cells were incubatedfor 1 hour at 4° C. before being washed again with FACS buffer and beingread on a FACSCalibur instrument. Monkey T lymphocyte population wasselected for analysis based on forward and side scatter as well aspositive staining for CD3 marker and negative staining for 7-AAD. Table6 shows the ability of purified anti-B7-H1 antibodies (1 μg/ml) to bindto activated human T cells as well as to activated cynomolgus monkey Tcells.

TABLE 6 Binding of purified anti-B7-H1 antibodies to stimulated human orcynomologus T-cells Human T cells Cyno T cells Ab ID (geo mean) (geomean) 2.9D10 49.0 106.8 2.14H9 53.0 106.3 3.15G8 48.0 92.8 2.20A8 41.080.7 2.7A4 49.0 103.9 3.18G1 45.0 94.6 IgG2 10.0 nd IgG4 10.0 17.6

Example 8: Inhibition of B7-H1/PD-1 Receptor-Ligand Binding

The ability of purified human anti-B7-H1 antibodies to inhibit thebinding of human PD-1/Fc protein to human B7-H expressed on surface ofES-2 cells (ATCC, catalog #CRL-1978) was evaluated. Briefly, 50000cells/well were plated in 50 μl of PBS into wells of a 384-well tissueculture plate (Corning Costar, catalog #3712). Next, 50 μl/well of adiluted monoclonal antibody was added at final concentration of 2.5,0.5, 0.1, 0.02, 0.004, 0.008, 0.00016 nM and plates were incubated at 4C for 1 hour. Cells were washed twice and 100 μl/well of biotinylatedhuman PD-1/Fc protein (10 ug/ml, R&D Systems, catalog #1086-PD) wasadded and plates were incubated at 4 C for 1 hour. Cells were washedonce and 100 μl/well of Cy5-conjugated streptavidin was added and plateswere incubated at 4 C for 15 min before being washed again with PBScontaining 2% FCS and being read on a FACSCalibur instrument. Some wellswere incubated with irrelevant human IgG2 and IgG4 monoclonal antibodieswith or without biotin-conjugated human PD-1/Fc to set minimal (0%) andmaximal (100%) level of B7-H1/PD-1 binding inhibition. The percentage ofthe inhibition in relation to the antibody concentration was analyzedusing curve fit tool (GraphPad Prism software) to calculate IC50 valuefor each antibody, which are shown in table 7.

TABLE 7 Inhibition of human PD1 binding to human B7-H1 expressing ES-2cell by purified anti-B7-H1 antibodies Ab ID IC50, nM 2.9D10 0.1092.14H9 0.083 3.15G8 0.148 2.20A8 0.198 2.7A4 0.077 3.18G1 0.140

Example 9: Determination of the Effects of Anti-B7-H1 Antibodies onProliferation of CD4 T Cells

It has been demonstrated that B7-H1 protein co-presented with anti-CD3antibody on beads inhibits CD3-mediated T cell activation (Freeman etal., J. Exp. Med., 2000, 192 (7): 1027-1034; Bennet et al., The Journalof Immunology, 2003, 170: 711-718). The ability of purified humanmonoclonal anti-B7-H1 antibodies to interfere with B7-H1-mediatedsuppression of T cell activation was determined as follows.

Briefly, 5×10(8) beads/ml of washed tosyl-activated Dynabeads M-450(Invitrogen, Cat #140.13) were coated with 50 ug/ml of mouse anti-humanCD3 antibody (BD Bioscience, Cat #555329) in 0.1 M sodium phosphatebuffer (pH 7.4-8.0) at 37° C. for 24 hours with shaking. 4×10(8) ofCD3-coated beads/ml were further coupled with recombinant human IgG1Fc(R&D Systems, cat #110-HG-100) at 160 ug/ml or with the recombinanthuman B7-H1/Fc protein (R&D Systems, Cat #156-B7-100) at 80 ug/mlcombined with the human IgG1Fc protein at 80 ug/ml (total concentration160 ug/ml) and incubated at 37° C. for 24 hours with shaking. The beadswere then incubated in PBS containing 0.05% bovine serum albumin at roomtemperature for 1 hour, washed four times in 0.1% BSA and 2 mM EDTA inPBS (pH7.4) and finally resuspended in RPMI1640 media containing 10% FBSat 5×10(7) beads/ml.

Peripheral blood monocytes were isolated from a leukopheresis pack usingFicoll-Paque Plus (GE Healthcare 17-1440-03) density gradientcentrifugation, resuspended in serum-free RPMI 1640 (Gibco 22400-089),and CD4+ T-cells were isolated from PBMC using Dynal CD4 NegativeIsolation Kit (Invitrogen, cat #113-37D) per manufacturer'sinstructions. 10 ul of coated beads were mixed 10 ul of dilutedanti-B7-H1 or control IgG2/4 antibody in a sample tube and incubated atRT for 3-4 hours on a shaker. Purified CD4+ T cells were plated at 10(5)cells/80 ul/well in 96-well plate (Corning, cat #3603) and bead-antibodymix was added at 20 ul/well to a total volume of 100 ul/well. T cellactivation in the absence of B7-H1 inhibitory effect was determinedusing beads coated with anti-CD3 antibody and the human IgG1Fc protein.Cells were cultured for 5 days and supernatants were harvested andanalyzed for IFN-γ release by using BD Human IFN-γ ELISA Kit II (BD Cat.No. 550612) per manufacturer's instructions. Cell proliferation wasmeasured on day 5 by the addition of 10 μl/well of AlamarBlue(Invitrogen DAL 1025). Cells were incubated for 5 hours, and thefluorescence was quantitated on a SpectraMax Gemini EM spectrophotometerwith an excitation wavelength of 545 nm and an emission wavelength of600 nm. Data are represented as the percentage of ODmax, where ODmax isthe average value obtained with T cells activated with anti-CD3/IgG1Fcbeads. See FIG. 1 .

Example 10: Determination of the Effects of Anti-B7-H1 Antibodies onActivation of CD4 T Cells in Dendritic Cell-T-Cell Mixed LymphocyteAssay

Enhancement of T cell activation by antibodies directed against B7-H1was determined in a dendritic cell-T-cell mixed lymphocyte (DCMLR)assay. Dendritic cells were generated from monocytic precursors asdescribed previously (Cuff Protoc Immunol. 2001 May; Chapter 7: Unit7.32). Peripheral blood monocytes were isolated from a leukopheresispack using Ficoll-Paque Plus (GE Healthcare 17-1440-03) density gradientcentrifugation, resuspended in serum-free RPMI 1640 (Gibco 22400-089),and allowed to adhere to T150 cell culture flasks (Corning 430825).After 1 hour at 37° C., the nonadherent cells were removed and the cellswere cultured in RPMI supplemented with 5% human serum (Invitrogen34005100). Cytokines were added at a final concentration of 2 ng/mlGM-CSF (BD Biosciences 550068) and 10 ng/ml IL-4 (BD Biosciences554605). Fresh media with cytokines was added every 2-3 days. At day 6of culture, cells were matured with 20 ng/ml of TNF-α (BD Biosciences554618) and allowed to incubate for 24 hours. Mature dendritic cellswere harvested, phenotyped, and frozen for later use.

CD4+ T-cells were isolated from PBMC using a magnetic isolation kit(Dynal 113.17) per manufacturer's instructions and cultured in a primaryMLR as described previously (J Immunol. 2003 Apr. 1; 170(7):3637-44).1.5E5 allogeneic CD4+ responding T-cells were cultured in 96 well-flatbottom microtiter plates (Costar 3595) with dendritic cells at aT-cell:dendritic cell ratio of 1:2.5. Dendritic cell preparations weretreated with 100 μg/ml of mitomycin C (Sigma M4287) prior to addition tococulture to prevent any proliferation from contaminating lymphocytes.Antibodies were added at various concentrations in a final volume of 200μl of RPMI+10% human serum. Thymidine incorporation was measured on Day5 by a 16-h pulse with [³H]thymidine (1 μci/well, Perkin-ElmerNET027001MC). Supernatants were harvested prior to radioactive labelingand analyzed for IFN-γ release by Luminex assay (BioRad 171-B11921) permanufacturer's instructions. Enhancement of T-cell proliferation byanti-B7-H1 antibodies from repeat experiments is shown in FIG. 2 .Corresponding IFN-γ release is shown in FIG. 3 .

Example 11: Structural Analysis of B7-H1 Antibodies

The heavy chain variable domain sequences and the light chain variabledomain sequence s of the antibodies were sequenced to determine theirDNA sequences. The complete sequence information for the anti-B7-H1antibodies is provided in the sequence listing with nucleotide and aminoacid sequences for each gamma and kappa or lambda chain combination. Thevariable heavy sequences were analyzed to determine the VH family andthe J-region sequence. The sequences were then translated to determinethe primary amino acid sequence and compared to the germline VH andJ-region sequences to assess somatic hypermutations.

Tables 8 and 9 are tables comparing the antibody heavy chain regions totheir cognate germline heavy chain region and the antibody light chainregions to their cognate germline light chain region. The amino acidnumbering is by numerical numbering.

Immunoglobulin genes undergo various modifications during maturation ofthe immune response, including recombination between V, D and J genesegments, isotype switching, and hypermutation in the variable regions.Recombination and somatic hypermutation are the foundation forgeneration of antibody diversity and affinity maturation, but they canalso generate sequence liabilities that may make commercial productionof such immunoglobulins as therapeutic agents difficult, or increase theimmunogenicity risk of the antibody. In general, mutations in CDRregions are likely to contribute to improved affinity and function,while mutations in framework regions may increase the risk ofimmunogenicity. This risk can be reduced by reverting frameworkmutations to germline, while ensuring that activity of the antibody isnot adversely impacted. Some structural liabilities may be generated bythe diversification processes, or they may exist within germlinesequences contributing to the heavy and light chain variable domains.Regardless of the source, it may be desirable to remove potentialstructural liabilities that may result in instability, aggregation,heterogeneity of product, or increased immunogenicity. Examples ofundesirable liabilities include unpaired cysteines (which may lead todisulfide bond scrambling, or variable sulfhydryl adduct formation),N-linked glycosylation sites (resulting in heterogeneity of structureand activity), as well as deamidation (e.g. NG, NS), isomerization (DG),oxidation (exposed methionine), and hydrolysis (DP) sites.

In order to reduce the risk of immunogenicity, and improvepharmaceutical properties of lead antibodies, it may be desirable toreduce the number of mutations from germline and/or remove structuralliabilities.

Thus, in one embodiment, where a particular antibody differs from itsrespective germline sequence at the amino acid level, the antibodysequence can be mutated back to the germline sequence. Such correctivemutations can occur at one, two, three or more positions, or acombination of any of the mutated positions, using standard molecularbiological techniques.

Tables 10-14 below illustrate the positions of such variations back togermline for mAb 2.9D10, 2.7A4 and 2.14H9. Each row represents a uniquecombination of germline and non-germline residues at the positionindicated by bold type. The position of the amino acid is representatedby numerical numbering.

In another embodiment, the invention includes replacing any structuralliabilities in the sequence that might affect the heterogeneity of theantibodies of the invention. Such liabilities include glycosylationsites, un-paired cysteines, surface exposed methinones, etc. To reducethe risk of such heterogeneity it is proposed that changes are made toremove one or more of such structural liabilities.

In one embodiment, it may be desirable to remove one or more consensusN-linked glycosylation sites from the antibody germline or antibodysequence. One skilled in the art would be readily able to identify sucha glycosylation site. Typically an N-linked glycosylation consensus sitesequence has the sequence of Asn-any AA-Ser or Thr where the middleamino acid cannot be a proline (Pro). In another example, unpairedcysteines can be replaced alone or in conjunction with other structuralchanges. An unpaired cysteine can be mutated to an appropriate aminoacid that has comparable side chain properties such as a serine.

As referred to herein, a sequence that is optimized is a sequence whichhas been mutated at one or more positions back to its germline sequenceor can be modified to remove one or more other liabilities such asstructural liabilities. An optimized sequence can also include asequence that has been mutated at one or more positions back to itsgermline sequence and which has also been further modified to remove oneor more structural liabilities.

TABLE 8 FR2 SEQ  CDR2 CDR3 FR4 SEQ ID NOS SEQ ID FR3 SEQ SEQ ID ID NOSCDR1 104, NOS 24, ID NOS NOS 113, 119, 119, FR1 SEQ SEQ ID NOS 104, 104,24, 24, 108, 108, 25, 114, 119, 119, Full ID NOS 87, 97, 98, 97, 104, 104, 44, 24, 108, 109, 45, 115, 120, 120, length 87, 87, 87,97, 97, 97, 104, 105, 14, 106, 108, 108, 15, 116, 119, 119, SEQ87, 87, 94, 99, 100, 105, 105, 4, 107, 108, 110, 5, 117, 119, 119, HeavyID 94, 95, 95, 101, 102, 105, 105 34, 107 111, 111,  35, 118  119 andchain V D J NO: 95 and 96 101 and 103 and 105 and 54 111 and 112 and 55119 Germline 81  EVQLVESGGG GFTFSSYWMS WVRQAPGK NIKQDGS RFTISRDNAKNSL###WFGEL WGQGTLVT LVQPGGSLRL GLEWVA EKYYVDS YLQMNSLRAEDTA #FDY VSS SCAASVKG VYYCAR 2.14H9 VH3-7 D3- JH4B 22 ---------- -----R---- --------------- ------------- EGG----- -------- 10 ---------- ------ -------------------- A--- --- ----- --- ------ Germline 82 EVQLVESGGGGFTFSSYWMS WVRQAPGK NIKQDGS RFTISRDNAKNSL #QL###YF WGQGTLVT LVQPGGSLRLGLEWVA EKYYVDS YLQMNSLRAEDTA DY VSS SCAAS VKG VYYCAR 3.15G8 VH3-7 D6-6JH4B 42 ---------- ---------- -------- ------G ------------- V--YSD---------- ---------- ------ ------- F------------ -- --- ----- --- ------Germline 83 EVQLVESGGG GFTFSSYWMS WVRQAPGK NIKQDGS RFTISRDNAKNSLDWNYYYYG WGQGTTVT LVQPGGSLRL GLEWVA EKYYVDS YLQMNSLRAEDTA MDV VSS SCAASVKG VYYCAR 2.9D10 VH3-7 D1-1 JH6B 12 ---------- ---------- --------------G ------------- ----G--D -------- ---------- ------ -Q------------------ --- --- ----- --- ------ Germline 84 EVQLVESGGG GFTFSSYSMNWVRQAPGK SISSSSS RFTISRDNAKNSL ###TAMV# WGQGTLVT LVQPGGSLRL GLEWVSYIYYADS YLQMNSLRAEDTA FDY VSS SCAAS VKG VYYCAR 2.7A4 VH3-21 D5-5 JH4B  2---------- -----T---- -------- -----GD ------------- DLV-S--A ------------------ ------ ------- F------K----- --- --- ----- --- ------Germline 85 EVQLVESGGG GFTFSSYAMS WVRQAPGK AISGSGG RFTISRDNAKNSL###YDSSG WGQGTLVT LVQPGGSLRL GLEWVS STYYADS YLQMNSLRAEDTA ##DY VSS SCAASVKG VYYCAK 2.20A8 VH3-23 D3- JH4B 32 ---------- -----N---- ----------R---- ------------- DLH----- -------- 22 ---------- ------ -------------------- YL-- ------ ----- --- ------ Germline 86 EVQLVESGGGGFTFSSYAMS WVRQAPGK AISGSGG RFTISRDNAKNSL ###GYSY# WGQGTLVT LVQPGGSLRLGLEWVS STYYADS YLQMNSLRAEDTA ##DY VSS SCAAS VKG VYYCAK 3.18G1 VH3-23D5-5 JH4B 52 ---------D ----N----- -------- T------ -------------VLV-PNN -------- ---------- ------ F-FS--- F-------V--S- GCW-- --------- --- --S---

TABLE 9 CDR2  CDR3 FR4 SEQ. FR1 SEQ FR2 SEQ ID SEQ ID SEQ ID ID NOSID NOS NOS 128, NOS 134, FR3 NOS 141, 147, 148, 121, 121, CDR1 128, 128,29, 134, SEQ. ID NOS 30, 142, 149, 149, FulL 121, 121, SEQ ID NOS129, 130, 19, 135, 136, 136, 136, 20, 143, 147, 147, length 122, 122,126, 28, 126, 131, 130, 49, 135, 136, 137, 138, 50, 144, 150, 150,  SEQ122, 122, 18, 48,  48, 130, 132, 39, 9, 137, 137, 139, 40, 145,151, 152,  Light ID 123, 124, 48, 38, 127, 132, 133 9, 59  139, 14010, 146 151 and Chain V J NO: 125 and 125 8, 58 and 58 and 133 and 59and 140 and 60 153 Germline 88 EIVLTQSPGTLS RASQSVSSSYLA WYQQKPGQAPRLGASSRAT GIPDRFSGSGSGTDF QQYGS##WT FGQGTKVE LSPGERATLSC LIYTLTISRLEPEDFAVY IK YC 2.14H9 A27 JK1 27 ------------ ----R------------------- D------ --------------- -----LP-- -----E-- ----------- ------------------ -- -- Germline 89 EIVLTQSPGTLS RASQSVSSSYLA WYQQKPGQAPRLGASSRAT GIPDRFSGSGSGTDF QQYGSS#FT FGPGTKVD LSPGERATLSC LIYTLTISRLEPEDFAVY IK YC 2.9D10 A27 JK3 17 ------------ --------N----F---------- -T----- --------------- ------I-- -------- ----------- --F--------------- -- -- Germline 90 DIQMTQSPSSVS RASQGISSWLA WYQQKPGKAPKLAASSLQS GVPSRFSGSGSGTDF QQANSFPPT FGQGTKVE ASVGDRVTITC LIYTLTISSLQPEDFATY IK YC 3.15G8 L5 JK1 47 ------------ ----------------S----- ---G--- --------------- --SH-L--- -------- ----------- ----------------L--- -- -- Germline 91 DIQMTQSPSSVS RASQGISSWLA WYQQKPGKAPKLAASSLQS GVPSRFSGSGSGTDF QQANSF##T FGGGTKVE ASVGDRVTITC LIYTLTISSLQPEDFATY IK YC 2.20A8 L5 JK4 37 ------------ ------R---------------- -I-R--- --------------- ------PL- -------- ----------- ------------------ -- -- Germline 92 SYELTQPPSVSV SGDALPKKYAY WYQQKSGQAPVLEDSKRPS GIPERFSGSSSGTMA YSTDSSGNH FGGGTKLT SPGQTARITC VIYTLTISGAQVEDEADY RV VL YC 2.7A4 V2-7 JL3  7 ------------ ------Q--VF------------ ------- --------------- ----R---- -----R-- ----A----- ------------------ -- -- -- Germline 93 SYVLTQPPSVSV GGNNIGSKSVHWYQQKPGQAPVL DDSDRPS GIPERFSGSNSGNTA QVWDSSSDH FGGGTKLT APGQTARITC VVYTLTISRVEAGDEADY VV VL YC 3.18G1 V2-14 JL2 57 ------------ ----------------------- ------- --------------- ------N-- -------- ---------- ------------------ -- -- --

TABLE 10 Exemplary Mutations of 2.7A4 Heavy Chain (SEQ ID NO: 2) toGermline at the Indicated Residue Number 31 55 56 80 87 S S S F K T S SF K S G S F K T G S F K S S D F K T S D F K S G D F K T G D F K S S S YK T S S Y K S G S Y K T G S Y K S S D Y K T S D Y K S G D Y K T G D Y KS S S F R T S S F R S G S F R T G S F R S S D F R T S D F R S G D F R TG D F R S S S Y R T S S Y R S G S Y R T G S Y R S S D Y R T S D Y R S GD Y R T G D Y R

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 2. In certainembodiments, SEQ ID NO.: 2 comprises any one of the combinations ofgermline and non-germline residues indicated by each row of Table 10. Insome embodiments, SEQ ID NO: 2 comprises any one, any two, any three,any four, any five, or all five of the germline residues as indicated inTable 10. In certain embodiments, SEQ ID NO.: 2 comprises any one of theunique combinations of germline and non-germline residues indicated byeach row of Table 10. In one specific example, the non germline sequenceis mutated back to germline at position 80 where F is changed to a Y andat position 87 where K is changed to an R. A specific example of such asequence is 2.7A4VHOPT as shown in Table 15.

TABLE 11 Exemplary Mutations of 2.7A4 Light Chain (SEQ ID NO: 7) toGermline at the Indicated Residue Number 17 29 32 33 104 T K A Y K A K AY K T Q A Y K A Q A Y K T K V Y K A K V Y K T Q V Y K A Q V Y K T K A FK A K A F K T Q A F K A Q A F K T K V F K A K V F K T Q V F K A Q V F KT K A Y R A K A Y R T Q A Y R A Q A Y R T K V Y R A K V Y R T Q V Y R AQ V Y R T K A F R A K A F R T Q A F R A Q A F R T K V F R A K V F R T QV F R A Q V F R T K A Y R A K A Y R T Q A Y R A Q A Y R T K V Y R A K VY R T Q V Y R A Q V Y R T K A F R A K A F R T Q A F R A Q A F R T K V FR A K V F R T Q V F R A Q V F R

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 7. In certainembodiments, SEQ ID NO.: 7 comprises any one of the combinations ofgermline and non-germline residues indicated by each row of Table 11. Insome embodiments, SEQ ID NO: 7 comprises any one, any two, any three,any four, any five, or all five of the germline residues as indicated inTable 11. In certain embodiments, SEQ ID NO.: 7 comprises any one of theunique combinations of germline and non-germline residues indicated byeach row of Table 11. Specific examples of 2.7A4 variable light domainwhich has been mutated to particular germline sequences include 2.7A4VLOPT (optimized where the non-germline sequence has been mutated froman A to a T at position 17 and a R to a K at position 104) as shown inTable 15.

TABLE 12 Exemplary Mutations of 2.9D10 Heavy Chain (SEQ ID NO: 12) toGermline at the Indicated Residue Number 56 58 G K S K G Q S Q

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 12. In certainembodiments, SEQ ID NO.: 12 comprises any one of the combinations ofgermline and non-germline residues indicated by each row of Table 12. Insome embodiments, SEQ ID NO: 12 comprises any one, any two, or all twoof the germline residues as indicated in Table 12. In certainembodiments, SEQ ID NO.: 12 comprises any one of the unique combinationsof germline and non-germline residues indicated by each row of Table 12.

TABLE 13 Exemplary Mutations of 2.9D10 Light Chain (SEQ ID NO: 17) toGermline at the Indicated Residue Number 32 37 50 52 S Y Y A N Y Y A S FY A N F Y A S Y F A N Y F A S F F A N F F A S Y Y T N Y Y T S F Y T N FY T S Y F T N Y F T S F F T N F F T S Y Y A N Y Y A S F Y A N F Y A S YF A N Y F A S F F A N F F A S Y Y T N Y Y T S F Y T N F Y T S Y F T N YF T S F F T N F F T

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 17. In certainembodiments, SEQ ID NO.: 17 comprises any one of the combinations ofgermline and non-germline residues indicated by each row of Table 13. Insome embodiments, SEQ ID NO: 17 comprises any one, any two, any three,any four, or all four of the germline residues as indicated in Table 7.In certain embodiments, SEQ ID NO.: 17 comprises any one of the uniquecombinations of germline and non-germline residues indicated by each rowof Table 13. SEQ ID NO: 17 can be altered or further altered by makingnongermlining changes to SEQ ID NO: 17. In one example, SEQ ID NO:17 canbe altered such that from a F to a Y at position 37 and from a F to a Yat position 50.

TABLE 14 Exemplary Mutations of 2.14H9 Light Chain (SEQ ID NO: 27) toGermline at the Indicated Residue Number 28 51 104 R G K S G K R D K S DK R G K S G K R D K S D K R G E S G E R D E S D E R G E S G E R D E S DE

In some embodiments of the invention, the targeted binding agent orantibody comprises a sequence comprising SEQ ID NO.: 27. In certainembodiments, SEQ ID NO.: 27 comprises any one of the combinations ofgermline and non-germline residues indicated by each row of Table 14. Insome embodiments, SEQ ID NO: 27 comprises any one, any two, any three,or all three of the germline residues as indicated in Table 14. Incertain embodiments, SEQ ID NO.: 27 comprises any one of the uniquecombinations of germline and non-germline residues indicated by each rowof Table 14. Specific examples of 2.7A4 variable light domain which hasbeen mutated to a particular germline sequence including 2.14H9OPT(optimized where the non-germline sequence has been mutated from an E toa K at position 104) as shown in Table 15.

The heavy chain of 2.14H9 can be changed at amino acid 31 of SEQ IDNO:22 from a R to a S.

TABLE 15 Full CDR1 SEQ FR2 CDR2 FR4 length FR1 SEQ ID NOS SEQ ID NOSSEQ ID NOS FR3 CDR3 SEQ ID SEQ ID ID NOS 153, 63, 68 105, 132 64, 69SEQ ID NOS 108, SEQ ID NOS NOS 119, NO: 123 and 121 and 78 and 128and 79 139 and 136 65,70 and 80 151 and 147 2.7A4 62 EVQLVESGGGL TYSMNWVRQAPGKGL SISSSGDYW RFTISRDNAKNSL DLVTSMVA WGQGTLV OPT VKPGGSLRLSC EWVSYADSVKG YLQVINSLRAEDT FDY TVSS Heavy AASGFTFS AVYYCAR Chain 2.7A4 67SYELTQPPSVSV SGDALPQK WYQQKSGQAP EDSKRFS GIPERFSGSSSGTM YSTDRSGNHFGGGTKLT OPT SPGQTARITC YVF VLVIY ATLTISGAQVEDE RV VL Light ADYYC Chain2.14H9 77 EIVLTQSPGTLS RASQRVSS WYQQKPGQAP DASSRAT GIPDRESGSGSGTDQQYGSLPW FGQGTKVE OPT LSPGERATLSC SYLA RLLIY FTLTBRLEPEDFA T IK LightVYYC ChainExamples of Specific Germlining

The amino acid sequences of the V_(H) and V_(L) domains of thenon-germlined (NG) anti-B7-H1 antibodies 2.7A4, 2.14H9 and 2.9D10 werealigned to the known human germline sequences in the VBASE database(Tomlinson, 1997; http://vbase.mrc-cpe.cam.ac.uk/), and the closestgermline was identified by sequence similarity. The closest germlinematch for the anti-B7-H1 antibodies is described in Tables 16 and 17.Without considering the Vernier residues (Foote & Winter, J Mol Biol.March 20:224(2):487-99, 1992), which were left unchanged, the positionsto be changed are described in Table 18.

TABLE 16 Anti-B7-H1 heavy chain closest V and J germline matches V J2.7A4 Heavy Chain VH3_(3-21) JH4b 2.9D10 Heavy Chain VH3_(3-07) JH6b2.14H9 Heavy Chain VH3_(3-07) JH4b

TABLE 17 Anti-B7-H1 light chain closest V and J germline matches V J2.7A4 Light Chain VL3_(3p) JL2 2.9D10 Light Chain VK3_(A27) JK3 2.14H9Light Chain VK3_(A27) JK1

Considering antibody 2.7A4 there are 2 changes in the frameworks of theV_(H) domain and 2 changes in the frameworks of the V_(L) domain. Theseresidues, located at Kabat number 79 and 83 (or residues 80 and 87 if bynumerical numbering) for the V_(H) domain and Kabat number 18 and 103(or residues 17 and 104 if by numerical numbering) for the V_(L) domain,were reverted to the human germline. See Table 18. For antibody 2.14H9there are no changes in the frameworks of the V_(H) domain and 1 changein the frameworks of the V_(L) domain. This residue, located at Kabatnumber 103 in the V_(L) domain (or 104 if residue calculated usingnumerical numbering), was reverted to human germline. See Table 18. Inantibody 2.9D10 there are no changes in the frameworks of the V_(H)domain and 2 changes in V_(L) domain, located at Kabat number at Kabatnumbers 36 and 49 (or numerical numbering residues 37 and 50). However,those 2 residues are located at Vernier position and have not beenmutated to not alter the CDR loop structures.

TABLE 18 Example of residues mutated during germlining for anti-B7-H1 Vdomains. Residues are numbering according to Kabat nomenclatureGermlined V domain Mutations 2.7A4VHOPT F79Y + K83R 2.7A4VLOPT A18T +R103K 2.14H9VLOPT E103K

Germlining of these amino acid residues was carried out using standardsite directed mutagenesis techniques with the appropriate mutagenicprimers. The germlined sequences have the prefix “OPT” after theantibody name, for example, 2.7A4VHOPT, 2.7VLOPT and 2.14H9VLOPT.Germlined IgG were then re-evaluated in the human B7-H1/hPD1 ligandinhibition assay to confirm there had not been a reduction in antibodyin vitro activity.

Example activities for germlined versus non-germlined anti-B7-H1antibodies are provided in FIG. 4 .

Example of Gene Synthesis and Reformatting as IgG1 and IgG1-TM

Clones were converted from scFv to IgG format by sub-cloning the V_(H)and V_(L) domains into vectors expressing whole antibody heavy and lightchains respectively. The V_(H) domains were cloned into the vectorpEU15.1 to express IgG1 or the vector pEU15.1-TM to express IgG-TMantibodies. Both vectors contain the human heavy chain constant domainsand regulatory elements to express whole IgG heavy chain in mammaliancells. The vector pEU15.1-TM is a modified pEU15.1 human IgG1 vector. Itwas engineered to introduce the three mutations; L234F and L235E in thehinge and P331S in the CH2 domain of the IgG molecule to eliminate itsability to trigger antibody-dependent cell-mediated cytotoxicity andcomplement-dependent cytotoxicity (Oganesyan V. et al. (2008), ActaCryst., D64: 700-704). Vector engineering was performed using standardsite directed mutagenesis techniques with the appropriate mutagenicprimers.

The V_(L) domains were cloned into pEU4.4 and pEU3.4 vectors for theexpression of the human lambda and kappa light chain constant domainsrespectively, with regulatory elements to express whole IgG light chainin mammalian cells. Vectors for the expression of heavy chains and lightchains were originally described in Vaughan et al. (Nature Biotechnology14(3):309-314, 1996). These vectors have been engineered simply byintroducing an OriP element.

To obtain IgGs, the heavy and light chain IgG expression vectors weretransfected into EBNA-HEK293 mammalian cells (Invitrogen R620-07). IgGswere expressed and secreted into the medium. Harvests were pooled andspun down prior to purification. The IgG was purified using Protein Achromatography using an AKTA Express purification system (GE Healthcare)previously sanitised to avoid any endotoxin contamination of the sample.Culture supernatants are loaded onto mL HiTrap™ MabSelectSure™ columns(GE Healthcare, 11-0034-93) and washed with 50 mM Tris-HCl pH 8.0, 250mM NaCl. Bound IgG was eluted from the column using 0.1 M Sodium Citrate(pH 3.0) and neutralised by the addition of 1M Tris-HCl (pH 9.0). Theeluted material was buffer exchanged into PBS using Nap10 columns(Amersham, 17-0854-02) and filtered before determining proteinconcentration and endotoxin levels. IgG concentration was determinedspectrophotometrically using an extinction coefficient based on theamino acid sequence of the IgG (Vaughan et al. supra). Endotoxin levelwas determined using the Endosafe PTS Portable Test System (CharlesRiver Laboratories) fitted with 1-0.1 EU/mL and 10-0.1 EU/mL LALcartridges (Charles River Laboratories, PT520). The purified IgG wereanalysed for degradation by SDS-PAGE.

Anti-B7-H1 antibodies in the IgG1-TM format were evaluated and comparedto the same antibodies but in the IgG1 format in the human B7-H1/hPD1ligand inhibition assay to confirm there had not been a reduction inantibody in vitro activity due to IgG isotype switching (FIG. 4 ).

Example 12: Human B7H1/FC Binding Human PD1/Fc—HTRF® Assay

The assay described is a homogenous TR-FRET assay using HTRF® assaytechnology requiring no wash steps. To a Costar 3676 microtitre plate 5μl/well of biotinylated PD1/Fc at 1 nM diluted into PBS was added. Thiswas followed with the addition of 5 μl/well streptavidin XL^(ent)(CisBio) at 4 nM diluted into assay buffer (PBS+0.1% BSA+0.8M KF). 5μl/well of a titration of sample material diluted in PBS was added torelevant wells. For the definition of total binding, 5 μl of PBS orrelevant sample buffer was added per well. To define non-specificbinding, an excess (600 nM) of unlabelled B7H1/Fc or PD1/Fc was used.The final process was the addition of 5 μl/well of cryptate labelledB7H1/Fc (cryptate label—CisBio, B7H1/Fc—RnD Systems) diluted 1:100 intoassay buffer. The assay plate was left for 3 hours at room temperaturebefore being read on a HTRF® compatible plate reader.

Example of IC50 determinations in the human PD1/human B7-H1 ligandinhibition assay for anti-B7-H1 antibodies are provided in Table 19. Allanti-human B7-H1 antibodies are in the IgG1-TM format.

TABLE 19 Example of IC50 determination (n = 2) in the human PD1/humanB7-H1 ligand inhibition assay for anti-B7-H1 IgG Arithmetic StandardAntibody mean Deviation 2.9D10 1.1E−10 3.6E−11 2.7A4OPT 1.6E−10 1.4E−102.14H9OPT 1.1E−10 7.5E−11

Example 13: Human B7H1/Fc Binding Human B7-1/Fc—HTRF® Assay

The assay described was a homogenous TR-FRET assay using HTRF® assaytechnology requiring no wash steps. To a Costar 3676 microtitre plate 5μl/well of biotinylated B7-1/Fc at 8 nM diluted into PBS was added. Thiswas followed with the addition of 5 μl/well streptavidin XL^(ent)(CisBio) at 20 nM diluted into assay buffer (PBS+0.1% BSA+0.8M KF). 5μl/well of a titration of sample material diluted in PBS was added torelevant wells. For the definition of total binding, 5 μl of PBS orrelevant sample buffer per well was added. To define non-specificbinding, an excess (200 nM) of unlabelled B7H1/Fc or B7-1/Fc was used.The final process was the addition of 5 μl/well of cryptate labelledB7H1/Fc (cryptate label—CisBio, B7H1/Fc—RnD Systems) diluted 1:100 intoassay buffer. The assay plate was left overnight at 4° C. before beingallowed to recover to room temperature and read on a HTRF® compatibleplate reader.

Example of IC50 determinations in the human B7-1/human B7-H1 ligandinhibition assay for anti-B7-H1 antibodies are provided in Table 20. Allanti-human B7-H1 antibodies are in the IgG1-TM format.

TABLE 20 Example of IC50 determination (n = 2) in the human B7-1/humanB7-H1 ligand inhibition assay for anti-B7-H1 IgG Arithmetic StandardAntibody mean Deviation 2.9D10 5.41E−11 1.61E−11 2.7A4OPT 5.31E−114.03E−13 2.14H9OPT 4.93E−11 1.50E−11

Example 14: Cross Reactivity of Anti-B7-H1 Antibodies with Other ImmuneCo-Modulatory Proteins

ELISAs were performed to determine the cross-reactivity of theanti-B7-H1 IgG-TM antibodies for other immune co-modulatory molecules.The ELISAs consisted of coating MaxiSorp plates (NUNC) at 4° C.overnight with 250 ng per well of the extracellular domain (ECD) ofhuman B7-H1 (R&D Systems, 156-B7), human PD-L2 (R&D Systems, 1224-PL),human B7-H2 (R&D Systems, 165-B7), human B7-H3 (R&D Systems, 1027-B3),human CD28 (R&D Systems, 342-CD), human CTLA-4 (R&D Systems, 325-CT) andhuman PD1 (R&D Systems, 1086-PD) followed by blocking the plates withPBS containing 3% dried milk powder at room temperature for 1 h. Murinecross reactivity was also investigated by coating the ECD of murineB7-H1 (R&D Systems, 1019-B7). Biotinylated anti-B7-H1 IgG1-TM diluted at100 nM in PBS containing 3% dried milk powder, were incubated at roomtemperature for 2h to allow binding. Bound biotinylated IgGs weredetected with europium N1-labelled streptavidin (Perkin Elmer, 1244-360)at 0.2 ug/mL. Control experiment demonstrating antigen coating to theNUNC plate was performed using the commercial antibodies mouse IgG2aanti-human B7-H1 (R&D Systems, MAB156), mouse IgG2b anti-human PD-L2(R&D Systems, MAB1224), mouse IgG2b anti-human B7-H2 (R&D Systems,MAB165), mouse IgG1 anti-human B7-H3 (R&D Systems, MAB1027), mouse IgG1anti-human CD28 (R&D Systems, MAB342), mouse IgG2a anti-human CTLA-4(Abcam, ab33320), mouse IgG2b anti-human PD1 (R&D Systems, MAB1086) andrat IgG2a anti-mouse B7-H1 (R&D Systems, MAB1019). Primary antibodieswere incubated at 5 ug/mL in PBS containing 3% dried milk powder for 2hat room temperature. Detection was carried out by incubating thesecondary antibody anti-mouse IgG Peroxidase conjugate (Sigma, A2554) oranti-rat IgG Peroxidase conjugate (Sigma, A5795) diluted 1:5000 in PBScontaining 3% dried milk powder for 1 h at room temperature andsubsequent addition of TMB (Sigma, T0440). All eight antigens could bedetected on the Maxisorp NUNC plate. Non-specific binding was determinedusing wells coated with the IgG1 isotype control at 5 ug/mL. Crossreactivities were calculated at a percent of the specific binding to theantigen relative to human B7-H1.

The cross-reactivity of the 3 anti-B7-H1 IgG-TM antibodies 2.7A4OPT,2.9D10 and 2.14H9OPT to the panel of eight immune co-modulatory antigenswere determined in triplicate. At 100 nM of antibody concentration, allthree anti-B7-H1 antibodies show no cross reactivity for any of theseven human immune co-modulatory molecules we tested (FIG. 5 ). TheIgG-TM anti-B7-H1 antibody 2.7A4OPT displays a measurable crossreactivity for murine B7-H1 with a signal level of 5.3% on averagecompared to 100% binding to the human B7-H1. The other two anti-B7-H1antibodies tested, 2.9D10 and 2.14H9OPT, do not show any crossreactivity for murine B7-H1 (FIG. 5 ).

Example 15: Affinity of Anti-B7-H1 Antibodies for Human and CynomologusB7-H1

The binding affinity and kinetic parameters of anti-B7-H1 antibodies inthe IgG-TM format to monomeric human and cynomologus B7-H1 weredetermined by surface plasmon resonance using a BIAcore T100 instrument(BIAcore, Uppsala, Sweden). In brief, experiments were performed at 25°C. using HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v/vsurfactant P20) as running buffer. IgGs were affinity captured on thesurface of a CM5 sensor chip (BIAcore) via protein G, which was aminecoupled onto the CM5 surface to achieve the density of ˜500 ResponseUnits (RU), according to the manufacturer's instructions(BIAapplications Handbook, BIAcore). Recombinant monomeric human orcynomologus B7-H1 FlagHis₁₀ extracellular domains (ECDs) were used asanalytes. Dilutions of B7-H1 ECD (200-3.12 nM) in the running bufferwere injected at a constant flow rate of 100 μl/min for 60 seconds. Allmeasurements were baseline corrected by subtracting the sensorgramobtained with the control (activated-deactivated) flow cell, as well asdouble referenced with blank (zero analyte concentration) injections.Data were analyzed using the T-100 BIAevaluation software package andfitted to a simple 1:1 Langmuir binding model with local R_(max) andbulk refractive index set to 0. Data were calculated from at least twoindependent experiments. Mass transport effects were limited by keepingthe level of affinity-captured IgGs below 250 RU. Sensorgrams obtainedwith all tested antibodies could be readily fitted onto themonoexponential 1:1 binding model giving excellent fits with Chi² valuesconsistently ≤0.3.

Affinity of anti-B7-H1 antibodies 2.7A4OPT and 2.14H9OPT for monomerichuman B7-H1 is 1 nM and 175 pM respectively (Table 21).

TABLE 21 Affinity and kinetic parameters analysis of anti- B7-H1antibodies for binding to human B7-H1 mAb/HUMAN B7H1 K_(d) (nM) k_(on)(M⁻¹ s⁻¹) k_(off) (s⁻¹) Reference Antibody #1 6.3 1.36 × 10⁶ 0.00862.7A4OPT 1 1.24 × 10⁶ 0.0012 2.14H9OPT 0.175   2 × 10⁶ 0.00035

Affinity of anti-B7-H1 antibodies 2.7A4OPT and 2.14H9OPT for monomericcynomologus B7-H1 is 835 pM and 367 pM respectively (Table 22). Thosetwo antibodies are strongly cross reactive for cynomologus B7-H1 asaffinities are very close to the ones for human B7-H1.

TABLE 22 Affinity and kinetic parameters analysis of anti- B7-H1antibodies for binding to cynomologus B7-H1 mAb/CYNO B7H1 K_(d) (nM)k_(on) (M⁻¹ s⁻¹) k_(off) (s⁻¹) Reference Antibody #1 4.64 1.36 × 10⁶0.0063 2.7A4OPT 0.835 1.31 × 10⁶ 0.0011 2.14H9OPT 0.367   1 × 10⁶0.00037

Example 16: Epitope Mapping of Anti-1B7-1H1 Antibodies

Epitope mapping was performed to identify human B7-H1 residues involvedin the binding of anti-B7-H1 antibodies. The structure of theextracellular domain of human B7H1 in complex with murine PD1 has beenpreviously described in the literature (Lin, D et al., 2008, Proc. Natl.Acad. Sci. USA, Vol. 105, p 3011-3016) and reveals that fourteen B17-H1residues are involved in the binding to PD1 (Table 23).

TABLE 23 Human B7-H1 residues involve in the binding to murine PD1 AminoAcid Position Phe 19 Thr 20 Asp 26 Ile 54 Tyr 56 Gln 66 Arg 113 Met 115Ser 117 Ala 121 Asp 122 Tyr 123 Lys 124 Arg 125

Anti-B7-H1 antibodies are competing for the binding of human B7-H1 tohuman PD1 (Example 5, 8 and 12) so they should interact with some ofthose 14 residues if we assume that there are negligible differencesbetween the B7H1 binding interface to human and mouse PD1 (62% aminoacid identity between the two species extracellular domain sequence).Single amino acid B7-H1 mutants will be generated for all the 14positions described in Table 23. Mutants will be tested for binding toanti-B7-H1 antibodies by phage ELISA and the ability to compete thebinding of anti-B7-H1 antibodies to human B7-H1 in HTRF® competitionassays. All anti-B7-H1 antibodies described are in the IgG-TM format.

Cloning of the Human B7-H1 Extracellular Domain Gene in Fusion withBacteriophage Gene III for Phage Display Expression

Gene coding for the extracellular domain of the human B7-H1 protein(Uniprot accession number Q9NZQ7, amino acid [19-238]) has beensynthetised externally by DNA2.0 Inc. and amplified by PCR using theprimers B7H1_FOR (5′-AATAATGGCCCAGCCGGCCATGGCCTTTACCGTGACGGTACCG-3′) andB7H1_REV (5′-AATAATGCGGCCGCCCTTTCGTTTGGGGGATGC-3′) to introduce the SfiI and Not I restriction sites at 5′ and 3′ ends respectively. PCRproduct has then been directionally cloned in the pCANTAB6 vector(McCafferty J. et al., 1994, Appl. Biochem. Biotechnol., Vol. 47, p157-173) using Sfi I and Not I restriction sites. E. coli strain TG1were transformed with the ligation and individual colonies were screenedby sequencing to identify a B7-H1 transformant named B7-H1_pCANTA6.

Generating and Identifying B7-H1 Mutants

B7-H1 mutants have been generated by saturation mutagenesis at all the14 residues of the PD1 interface using fully randomised NNS primers(Table 24) and the plasmid B7-H1_pCANTA6 as DNA template. Mutagenesiswas performed with the Stratagene's QuickChange Multi Site-DirectedMutagenesis Kit (Catalog #200513) according to the manufacturer'sinstructions. Mutagenic reactions were used to transform E. coli strainTG1 and individual colonies were screened by sequencing to identifyB7-H1 variants. A total of 252 variants were identified among the 280possible (20 amino acids time 14 positions) and cherry picked in 396-wells culture plates.

TABLE 24 Mutagenenie primers used to generate B7-H1  variantsPrimer Name Sequence B7HI_SDM_FI9_f 5′-GCCGGCCATGGCCNNSACCGTGACGGTACCGAAAG-3′ B7H1_SDM_T20_f 5′-GCCGGCCATGGCCTTTNNSGTGACGGTACCGAA AG-3′B7H1_SDM_D26_f 5′-CCGTGACGGTACCGAAANNSTTGTATGTGGTGG AGTATGG-3′B7H1_SDM_I54_f 5′-GGATCTTGCTGCCCTTNNSGTCTACTGGGAAAT GGAGGACAAG-3′B7H1_SDM_Y56_f 5′-GGATCTTGCTGCCCTTATCGTCNNSTGGGAAAT GGAGGACAAG-3′B7H1_SDM_Q66_f 5′-GGAGGACAAGAACATCATCNNSTTTGTCCATGG AGAGGAGG-3′B7H1_SDM_R113_f 5′-ATGCCGGGGTCTACNNSTGTATGATCTCTTACG GCGG-3′B7H1_SDM_M115_f 5′-ATGCCGGGGTCTACCGCTGTNNSATCTCTTACG GCGG-3′B7H1_SDM_S117_f 5′-CTACCGCTGTATGATCNNSTACGGCGGTGCCG- 3′ B7H1_SDM_A121_f5′-GATCTCTTACGGCGGTNNSGATTACAAACGGAT AACC-3′ B7H1_SDM_D122_f5′-GATCTCTTACGGCGGTGCCNNSTACAAACCGAT AACC-3′ B7H1_SDM_Y123_f5′-CGGCGGTGCCGATNNSAAACGGATAACCGTAAA GG-3′ B7H1_SDM_K124_f5′-CGGCGGTGCCGATTACNNSCGGATAACCGTAAA GG-3′ B7H1_SDM_R125_f5′-GCGGTGCCGATTACAAANNSATAACCGTAAAGG TAAACG-3′Phage ELISA of B7-H1 Mutants for Binding to Anti-B?-H1 Antibodies

Binding of the B7-H1 mutants to anti-B7-H1 antibodies 2.14H9OPT,2.7A4OPT or Reference Ab #1 have been assessed by phage ELISA afterassuring that the B7-H1 extracellular domain in fusion with gene IIIprotein could be displayed at the phage surface. Cherry picked TG1cultures were grown and superinfected with M13K07 helper phage toproduce phage particles displaying B7-H1 mutants at their surface. Phagesupernatants were blocked in PBS+3% skimmed milk and incubated in NUNCMaxiSorb plates previously coated overnight with 1 ug/mL 2.14H9OPT,2.7A4OPT or Reference antibody #1 in PBS and blocked with PBS+3% skimmedmilk. Bound phages were detected using streptavidin coupled witheuropium (Perkin Elmer) after incubation with a biotinylated anti-M13secondary antibody (Progen).

Among the 14 human B7-H1 residues located at the PD1 interface, four arenot involved in the binding to any of the three tested anti-B7-H1antibodies (Asp26, Tyr56, Glu66 and Lys124). Their replacements by analanine or a glycine do not significantly affect the binding signal.Based on the phage ELISA data, a total of 28 mutants representative of 2to 3 key changes for each of the 10 others positions have been selectedto confirm their binding profile but using purified proteins.

Biochemical Competition Assays

Extracellular domain of human B7-H1 wild-type and mutants were expressedin bacteria and purified by affinity chromatography as previouslydescribed (Bannister D. et al., 2006, Biotechnology and bioengineering,94, 931-937).

The HTRF® competition assays measured the binding of anti-B7-H1 antibodyto HIS FLAG tagged B7-H1. Titration of non tagged B7-H1 samples,prepared as described above, will compete with HIS FLAG tagged B7-H1 forbinding to anti-B7-H1 antibody, leading to a reduction in assay signal.The antibodies 2.14H9OPT, 2.7A4OPT and Reference antibody #1 were usedto establish competition assays for characterising the relative bindingof purified wild-type or mutants B7-H1. This will confirm which B7-H1residues are required for antibody binding. 10 μl of B7-H1 sample wasadded to a 384 well low volume assay plate (Corning 3673). This wasfollowed by the addition of 5 μL of either 0.29 nM 2.14H9OPT, or 1.15 nM2.7A4OPT, or 1.15 nM Reference antibody #1 conjugated to DyLight649according to manufacturer's instructions (ThermoFisher, 53051) and 5 μLof a mixed solution of 0.43 nM anti-FLAG cryptate (Cisbio International,61FG2KLB) and 1.25 nM HIS FLAG tagged human B7-H1. Assay plates wereincubated for 4 hours at room temperature prior to reading time resolvedfluorescence at 620 nm and 665 nm emission wavelengths using an EnVisionplate reader (Perkin Elmer).

Three additional B7-H1 residues (Ile54, Ser17 and Ala121) are notinvolved in the binding to 2.14H9OPT and 2.7A4OPT as IC₅₀ of B7-H1mutants for those residues are similar or marginally modified comparedto wild-type B7-H1. Competition data to the 3 anti-B7-H1 antibodies forwild-type and all the other B7-H1 mutants are summarised in the Table25.

TABLE 25 IC₅₀ in nM in the human B7-H1 competition assays for binding toanti-B7-H1 antibodies Competition assay IC₅₀ in nM 2.14H9OPT 2.7A4OPTReference B7-H1 sample IgG1-TM IgG1-TM Ab#1 wild-type B7H1 0.76 2.791.89 B7H1_F19A 0.56 No inhibition 27.6 B7H1_F19G 0.56 No inhibition 15.3B7H1_F19S 0.66 No inhibition 21.6 B7H1_T20A 0.45 No inhibition 2.3B7H1_T20V 0.64 No inhibition 1.9 B7H1_T20D 0.83 No inhibition 1.9B7H1_R113A No inhibition 4.6 172 B7H1_R113Y No inhibition No inhibition3.9 B7H1_R113L No inhibition No inhibition 2 B7H1_M115A 3.9 6.9 86B7H1_M115G 30.4 4.6 No inhibition B7H1_M115D No inhibition 5.8 Noinhibition B7H1_D122E 29.6 No inhibition 35.2 B7H1_D122N 15.5 Noinhibition 2.5 B7H1_Y123A 232 1.77 No inhibition B7H1_Y123F 0.5 2.21 Noinhibition B7H1_Y123T 427 2.07 No inhibition B7H1_R125A No inhibition 311.7 B7H1_R125Q No inhibition 2.4 18.4 B7H1_R125S No inhibition 2.7 9.7

Arg113 and Arg125 are strongly involved in the binding to 2.14H9OPT.Replacement by an Ala or other amino acids (Tyr or Leu at position 113and Gln or Ser at position 125) lead to a total lost of binding to thatantibody. Binding profile of those B7-H1 mutants to 2.7A4OPT orReference antibody #1 is similar to the wild-type B7-H1. This shows thatthe lost of binding is not due to a general structural modification ofB7-H1 as for instance an unfolded protein but to a direct involvement ofthose residues with 2.14H9OPT binding. Those data also demonstrate that2.14H9OPT binding epitope is different from 2.7A4OPT and Referenceantibody #1 epitopes. Met115, Asp122 and Tyr123 are also involved in thebinding to 2.14H9OPT but to a lesser extend. Replacement of Met115 by anAla does not affect the binding to 2.14H9OPT or 2.7A4OPT but replacementby an Asn leads to a total lost of binding activity to 2.14H9OPT but notto 2.7A4OPT. In a similar way, replacement of Asp122 by an Asn isaffecting the binding to 2.14H9OPT but not to Reference antibody #1.Mutating Tyr123 by an Ala or a Thr greatly modify the binding profile to2.14H9OPT but not to 2.7A4OPT. Interestingly, a replacement by a Phedoes not change the binding to 2.14H9OPT suggesting that the hydroxylgroup of the tyrosine 123 is not involved in the binding interaction.

Phe19, Thr20 and Asp122 are strongly involved in the binding to2.7A4OPT. All B7-H1 mutants at those positions have no binding to thatantibody but do bind 2.14H9OPT or Reference antibody #1 in a similar wayto wild-type B7-H1, excepted B7H1_D122E mutant which binds those 2antibodies less efficiently. Those three residues are specifics to the2.7A4OPT mAb binding epitope and are not shared by the 2.14H9OPT or theReference antibody #1 epitopes. Arg113 is also involved in the 2.7A4OPTepitope but to a lesser extend. Replacement by an Ala does not affectthe binding to that antibody but a Tyr or Leu mutations lead to a totallost of binding. Those mutations at position 113 do not affect thebinding to the Reference antibody #1 showing that B7-H1 mutants arestill in a correct conformation. It is possible that the replacement bya bulky amino acid as a Tyr may introduce some steric hindrances. Thoseresults suggest that the Arg113 is not directly involved in the contactto the 2.7A4OPT antibody but is very close to the actual bindingepitope.

This example demonstrates that the three anti-B7-H1 antibodies2.14H9OPT, 2.7A4OPT and Reference antibody #1 have distinct bindingepitopes at the B7-H1 interface with PD1. Furthermore, it is possiblethat those antibodies may also bind to other human B7-H1 residues butwhich are not located at the PD1 interface.

Example 17. Determination of the Effects of Anti-B7-H1 Antibodies on aMemory T-Cell Response to Sub-Optimal Concentrations of a Recall Antigen

B7-H1 interaction with PD-1 has been shown to inhibit antigen specificT-cell responses. In order to assess the effect of anti-B7-H1 antibodieson this inhibition a sub-optimal antigen recall assay was performed.

Peripheral blood monocytes (PBMCs) were isolated from human blood buffycoats using Ficoll-Paque Plus (GE Healthcare 17-1440-03) densitygradient centrifugation as per manufacturers instructions. Followingisolation cells were resuspended in RPMI 1640 Glutamax I media (GlBCO,□161870) supplemented with 1% pen/strep (GIBCO, □15140) and 4% Human ABSerum (Invitrogen □34005), and then subsequently cultured in 96 well,round bottom tissue culture plates (Corning, □13799) at 37° C., 5% CO2,with or without 0.1 μg/mL tetanus toxoid (Calbiochem □582231) at adensity of 1×105 cells per well. After three days of culture, anti-B7-H1antibodies in the IgG1-TM format or isotype control were added at theindicated concentrations and cultures returned to 37° C. for a further 2days, at which point supernatants were harvested and analysed by DELFIAfor levels of interferon-. Enhancement of interferon-γ release byanti-B7-H1 antibodies is shown in FIG. 6 .

Anti-B7-H1 antibodies 2.9D10, 2.7A4OPT and 2.14H9OPT are able toincrease the release of Interferon-γ. This data confirms the ability of2.9D10, 2.7A4OPT and 2.14H9OPT to enhance antigen specific T-cellresponses.

Example 18. Determination of the Effects of Anti-B7-H1 Antibodies on aMemory T-Cell Response to Optimal Concentrations of a Recall Antigen

B7-H1 has been suggested to have potential inhibitory signallingproperties. The potential for anti-B7-H1 antibodies to act as agonistthat might drive such inhibitory signalling was tested by examiningtheir ability to inhibit an antigen recall response.

Peripheral blood monocytes (PBMCs) were isolated from a human bloodbuffy coats using Ficoll-Paque Plus (GE Healthcare 17-1440-03) densitygradient centrifugation as per manufacturers instructions. Followingisolation cells were resuspended in RPMI 1640 Glutamax I media (GIBCO,□61870) supplemented with 1% pen/strep (GIBCO, □15140) and 4% Human ABSerum (Invitrogen □34005), and then subsequently cultured, at a densityof 1×105 cells per well, in 96 well, round bottom tissue culture plates(Corning, □73799) at 370 C, 5% CO2, together with 5 μg/mL tetanus toxoid(Calbiochem □582231) and in the presence or absence of varyingconcentrations of anti-B7-H1 antibodies in the IgG1-TM format or isotypecontrol. An antibody against a T-cell co-receptor has been used aspositive control. Following 5 days of culture cells were pulsed with 0.5μCi/well titiated thymidine for approximately 16 hours in order toassess proliferative activity.

No inhibitory effects were observed with 2.9D10, 2.7A4 and 2.14H9anti-B7-H1 antibodies, in contrast to positive control, as shown in FIG.7 . This suggests that 2.9D10, 2.7A4 and 2.14H9 antibodies are pureantagonists, without any agonistic activity.

Example 19. Tumor Growth Inhibition Activity of Anti-B7-H1 Antibodies

The in-vivo activity of anti-human B7-H1 antibodies was investigated inxenograft mouse models using immunocompromised NOD/SCID (non-obesediabetic/severe combined immunodeficiency) mice. The mice were engraftedsubcutaneously (SC) with human cancer cell lines expressing human B7-H1and human CD4+ and CD8+ T cells that were isolated from peripheral bloodmononuclear cells of healthy donors and cultured to enrich foralloreactive effector T cells. Intraperiteneal (IP) doses of anti-humanB7-H1 antibodies were given to mice inoculated with the human pancreaticcancer cell line HPAC or the human melanoma cell line A375. Effect ofthe antibodies was observed on tumor growth until a 2000 mm³ tumorvolume or gross tumor necrosis.

To generate CD4+ and CD8+ T cell lines, human PBMC's from healthy donorswere enriched for CD4+ or CD8+ T cells by the addition of 1 mLRosetteSep T cell enrichment product per 20 mL of whole blood. This wasfollowed by a 20-minute incubation and subsequent isolation by densitygradient centrifugation using RosetteSep DM-L density medium. Aftercentrifugation, the cells were washed with PBS and resuspended in RPMI1640 medium supplemented with 10% FBS. Enriched CD4+ and CD8+ T cellswere cultured separately for 7-10 days in medium supplemented withrhIL-2 and each combined with mitomyosin C treated A375 or HPAC cells. Tcells were collected and separately cultured again for 7-10 days inmedium supplemented with rhIL-2 and combined with mitomyosin C treatedA375 or HPAC cells. CD4+ and CD8+ T cells were collected and combined ata 1:1 ratio.

A375 and HPAC cancer cell lines and PBMC enriched for CD4+ and CD8+ Tcells were mixed immediately before subcutaneous (SC) administration atthe indicated effector-to-target (E:T) ratios. The inoculation number ofcells for each cancer cell line was predetermined by empirical tumorforming dose studies; in general, 2.5×10⁶ cells in a total volume of 0.2mL were engrafted into each animal.

Six animals were assigned to each experimental group. The animalsreceived an isotype control human IgG2a or IgG1OPT (also referred toherein as “IgG1TM”) antibody or the anti-B7-H1 antibodies 2.14H9 IgG2a,2.14H9OPT, 2.7A4OPT or Reference antibody #1 in the IgG1OPT format. Atotal of eight independent experiments have been carried out and studydesigns for each experiment are presented in Tables 26-33. Describedanti-B7-H1 antibodies are in the IgG1OPT format except where specified.

The first dose (200 μL) of test article was administered IP 1 hour afterengraftment of cancer/effector T cells; the animals received up to 4additional doses of the test article on study days 3, 5, 8 and/or 10. Asa positive control to enhance alloreactivity in some studies, rhIL-2 wasadministered IP 1 hour after engrafiment of cancer/effector T cells; theanimals received 4 additional daily doses of rhIL-2 for 4 consecutivedays. The formation of tumor was observed in each animal one or twotimes a week. Tumors were measured by caliper; tumor volumes (V) werecalculated using the following formula:V(mm³)=0.5(length(mm)×width(mm)×width(mm)/2).

For each group, the results are reported as the arithmetic mean.Anticancer effect, expressed as a percent tumor growth inhibition (TGI),was calculated by the following method:% TGI=[1−(mean tumor V of treatment group)+(mean tumor V of controlgroup)]×100.

In Study 1, anti-B7-H1 antibodies 2.14H9 IgG2a and 2.7A4OPTsignificantly inhibited the growth of HPAC (pancreas) cancer cells atday 30 by up to 61% and 50% respectively as compared to the isotypecontrol group (FIG. 8 and Table 26).

TABLE 26 Study 1 - Treatment groups and percent Tumor Growth Inhibitionin mice engrafted with HPAC cancer cells following intravenousadministration of anti-B7-H1 antibodies Dose % TGI at Group Test Article(mg/kg/mouse) day 30 1 None NA NA 2 None NA NA 3 rhIL-2 10⁵ U/dose NA 4Isotype human IgG2a 20 NA 5 Isotype human IgG1OPT 20 NA 6 2.14H9 IgG2a20 50 7 2.14H9 IgG2a 10 22 8 2.14H9 IgG2a 1 56 9 2.14H9 IgG2a 0.1 61 102.7A4OPT 20 <1 11 2.7A4OPT 10 30 12 2.7A4OPT 1 50 13 2.7A4OPT 0.1 15 NA= not applicable

In Study 2, anti-1B7-1 antibodies 2.14H9OPT and 2.7A4OPT inhibited thegrowth of HPAC (pancreas) cancer cells at day 39 by upto 70% and 68⁰%respectively as compared to the isotype control group (FIG. 9 and Table27).

TABLE 27 Study 2 - Treatment groups and percent Tumor Growth Inhibitionin mice engrafted with HPAC cancer cells following intravenousadministration of anti-B7-H1 antibodies Dose % TGI at Group Test Article(mg/kg/mouse) day 39 1 None NA NA 2 None NA NA 3 rhIL-2 10⁵ U/dose NA 4Isotype human IgG1OPT 20 NA 5 2.14H9OPT 20 67 6 2.14H9OPT 10 28 72.14H9OPT 1 63 8 2.14H9OPT 0.1 70 9 2.7A4OPT 20 54 10 2.7A4OPT 10 61 112.7A4OPT 1 50 12 2.7A4OPT 0.1 68 NA = not applicable

In Study 3, anti-17-1H1 antibody 2.141H9OPT significantly inhibited thegrowth of HPAC (pancreas) cancer cells at day 30 by upto 60% as comparedto the isotype control group (FIG. 10 and Table 28).

TABLE 28 Study 3 - Treatment groups and percent Tumor Growth Inhibitionin mice engrafted with HPAC cancer cells following intravenousadministration of anti-B7-H1 antibodies Dose % TGI at Group Test Article(mg/kg/mouse) day 30 1 None NA NA 2 None NA NA 3 rhIL-2 10⁵ U/dose NA 4Isotype human IgG1OPT 5 NA 5 Reference antibody#1 5 37 6 2.14H9OPT 5 607 2.14H9OPT 1 57 8 2.14H9OPT 0.1 57 9 2.14H9OPT 0.01 26 NA = notapplicable

In Study 4 anti-07-1 antibody 249OPT significantly inhibited the growthof HPAC (pancreas) cancer cells at day 22 by upto 74% as compared to theisotype control group (FIG. 11 and Table 29).

TABLE 29 Study 4 - Treatment groups and percent Tumor Growth Inhibitionin mice engrafted with HPAC cancer cells following intravenousadministration of anti-B7-H1 antibodies Dose % TGI at Group Test Article(mg/kg/mouse) day 22 1 None NA NA 2 None NA NA 3 Human IgG1OPT 5 NA 42.14 H9OPT 5 74 5 2.14 H9OPT 1 55 6 2.14 H9OPT 0.1 26 7 2.14 H9OPT 0.0121

In Study 5, IP administration of 2.141H9 IgG2a or 2.7OPT anti-137-1H1antibodies in the A375 (melanoma) xenograft model also significantlyinhibited tumor growth at day 29 by as much as 64% and 61% respectivelyas compared to the isotype control group (FIG. 12 and Table 30).

TABLE 30 Study-5. Treatment groups and percent Tumor Growth Inhibitionin mice engrafted with A375 cancer cells following intravenousadministration of anti-B7-H1 antibodies Dose % TGI at Group Test Article(mg/kg/mouse) day 29 1 None NA NA 2 rhIL-2 10⁵ U/dose NA 3 Isotype humanIgG2a 10 NA 4 Isotype human IgG2a 1 NA 5 Isotype human IgG2a 0.1 NA 6Isotype human IgG1OPT 10 NA 7 Isotype human IgG1OPT 1 NA 8 Isotype humanIgG1OPT 0.1 NA 9 2.14H9 IgG2a 10 54 10 2.14H9 IgG2a 1 37 11 2.14H9 IgG2a0.1 64 12 2.7A4OPT 10 21 13 2.7A4OPT 1 61 14 2.7A4OPT 0.1 55 NA = notapplicable

In Study 6, anti-B7-H1 antibody 2.14H9OPT significantly inhibited thegrowth of A375 (melanoma) cancer cells when combined with T cells at day25 by up to 77% as compared to the isotype control group (FIG. 13 andTable 31).

TABLE 31 Study-6. Treatment groups and percent Tumor Growth Inhibitionin mice engrafted with A375 cancer cells following intravenousadministration of anti-B7-H1 antibodies Dose % TGI at Group Test Article(mg/kg/mouse) day 25 1 None NA NA 2 None NA NA 3 Human IgG1OPT 5 NA 4Human IgG1OPT; no T cells 5 NA 5 2.14 H9OPT 5 67 6 2.14 H9OPT 1 77 72.14 H9OPT 0.1 72 8 2.14 H9OPT; no T cells 1 13 9 2.14 H9OPT; no T cells0.1 25 NA = not applicable

In Study 7, anti-B7-H1 antibody 2.14H9OPT significantly inhibited thegrowth of A375 (melanoma) cancer cells when combined with T cells at day25 by up to 82% as compared to the isotype control group (FIG. 14 andTable 32).

TABLE 32 Study-7. Treatment groups and percent Tumor Growth Inhibitionin mice engrafted with A375 cancer cells following intravenousadministration of anti-B7-H1 antibodies Dose % TGI at Group Test Article(mg/kg/mouse) day 25 1 None NA NA 2 None NA NA 3 Human IgG1OPT 5 NA 4Human IgG1OPT; no T cells 5 NA 5 2.14 H9OPT 5 55 6 2.14 H9OPT 1 82 72.14 H9OPT 0.1 72 8 2.14 H9OPT; no T cells 1 36 9 2.14 H9OPT; no T cells0.1 27 NA = not applicable

In Study 8, anti-B7-H1 antibody 2.14H9OPT significantly inhibited thegrowth of A375 (melanoma) cancer cells when combined with T cells at day25 by up to 93% as compared to the isotype control group (FIG. 15 andTable 33).

TABLE 33 Study-8. Treatment groups and percent Tumor Growth Inhibitionin mice engrafted with A375 cancer cells following intravenousadministration of anti-B7-H1 antibodies Dose Dose Schedule % TGI atGroup Test Article (mg/kg/mouse) (Study Day) day 25 1 None NA NA NA 2None NA NA NA 3 Human IgG1OPT 1 1, 3, 5, 8, 10 NA 4 2.14 H9OPT 1 1 93 52.14 H9OPT 1 1, 5 91 6 2.14 H9OPT 1 1, 10 93 7 2.14 H9OPT; 1 1, 5, 10 66no T cells 8 2.14 H9OPT; 1 1, 3, 5, 8, 10 64 no T cells NA = notapplicable

These results demonstrated that the anti-human B7-H1 antibodies 2.14H9IgG2a, 2.14H9OPT and 2.7A4OPT have potent in vivo anticancer activity inxenograft mouse models of human cancers and provide evidence that thoseanti-human B7-H1 antibodies can have activity as a single-agent therapyfor the treatment of patients with cancers expressing B7-H1.

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety. U.S. provisional patent application Ser.No. 61/264,061 filed Nov. 24, 2009, the figures and the sequencestherein are hereby incorporated by reference in its entirety.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The foregoingdescription and Examples detail certain preferred embodiments of theinvention and describes the best mode contemplated by the inventors. Itwill be appreciated, however, that no matter how detailed the foregoingmay appear in text, the invention may be practiced in many ways and theinvention should be construed in accordance with the appended claims andany equivalents thereof.

The invention claimed is:
 1. An isolated antibody or fragment thereofthat specifically binds to B7-H1, wherein the antibody or fragmentthereof comprises: a VH CDR1 consisting of the amino acid sequence ofSEQ ID NO: 3, a VH CDR2 consisting of the amino acid sequence of SEQ IDNO: 4, a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 5,a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 8, a VLCDR2 consisting of the amino acid sequence of SEQ ID NO: 9, and a VLCDR3 consisting of the amino acid sequence of SEQ ID NO: 10; or a VHCDR1 consisting of the amino acid sequence of SEQ ID NO: 63, a VH CDR2consisting of the amino acid sequence of SEQ ID NO: 64, a VH CDR3consisting of the amino acid sequence of SEQ ID NO: 65, a VL CDR1consisting of the amino acid sequence of SEQ ID NO: 68, a VL CDR2consisting of the amino acid sequence of SEQ ID NO: 69, and a VL CDR3consisting of the amino acid sequence of SEQ ID NO:
 70. 2. The antibodyor fragment thereof of claim 1, wherein the antibody is a monoclonalantibody.
 3. The antibody or fragment thereof of claim 2, wherein theantibody is a fully human monoclonal antibody.
 4. The antibody orfragment thereof according to any one of the preceding claims, whereinthe antibody or fragment thereof comprises a heavy chain variable domainconsisting of the amino acid sequence of SEQ ID NO: 2 and a light chainvariable domain consisting of the amino acid sequence of SEQ ID NO: 7;or a heavy chain variable domain consisting of the amino acid sequenceof SEQ ID NO: 62 and a light chain variable domain consisting of theamino acid sequence of SEQ ID NO:
 67. 5. The antibody or fragmentthereof of claim 4, wherein the fragment is a binding fragment selectedfrom the group consisting of a Fab, Fab′, F(ab′)2, Fv and dAb fragment.6. The antibody or fragment thereof of claim 4, wherein the Fc region ofthe antibody is characterized by the residues of 234F, 235E, and 331S,as numbered by the EU index as set forth in Kabat.
 7. A nucleic acidmolecule encoding the antibody of claim
 1. 8. An isolated host celltransfected with a vector comprising the nucleic acid molecule of claim7.
 9. An antibody produced by a method comprising, culturing the hostcell of claim 8, expressing an antibody encoded by the nucleic acidmolecule of claim 7, and isolating the antibody from the culture.
 10. Acomposition comprising the antibody of claim
 1. 11. A pharmaceuticalcomposition comprising the antibody of claim 1 and pharmaceuticallyacceptable carrier.